Method for enzymatic production of GLP-2(1-33) and GLP-2(1-34) peptides

ABSTRACT

The invention provides methods for making peptides from a polypeptide containing at least one copy of the peptide using clostripain to excise the peptide from the polypeptide. The methods enable the use of a single, highly efficient enzymatic cleavage to produce any desired peptide sequence.

This application is a divisional of U.S. patent application Ser. No.10/997,065, now U.S. Pat. No. 7,781,567, which was a continuation under35 U.S.C. 111(a) of PCT/US03/16649, filed on May 23, 2003 and publishedon Dec. 4, 2003 as WO 03/099854 A2, which claims priority under 35U.S.C. 119(e) of U.S. Provisional Application No. 60/383,359 and60/383,468, both filed on May 24, 2002, which applications andpublication are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Although bioactive peptides can be produced chemically by a variety ofsynthesis strategies, recombinant technology offers the potential forinexpensive, large-scale production of peptides without the use oforganic solvents, highly reactive reagents or potentially toxicchemicals. However, expression of short peptides in Escherichia coli andother microbial systems can sometimes be problematic. For example, shortpeptides are often degraded by the proteolytic and metabolic enzymespresent in microbial host cells. Use of a fusion protein to carry thepeptide of interest may help avoid cellular degradation processesbecause the fusion protein is large enough to protect the peptide fromproteolytic cleavage. Moreover, certain fusion proteins can direct thepeptide to specific cellular compartments, i.e., cytoplasm, periplasm,inclusion bodies or media, thereby helping to avoid cellular degradationprocesses. However, while use of a fusion protein may solve certainproblems, cleavage and purification of the peptide away from the fusionprotein can give rise to a whole new set of problems.

Preparation of a peptide from a fusion protein in pure form requiresthat the peptide be released and recovered from the fusion protein bysome mechanism. In many cases, the peptide of interest forms only asmall portion of the fusion protein. For example, many peptidyl moietiesare fused with β-galactosidase that has a molecular weight of about100,000 daltons. A peptide with a molecular weight of about 3000 daltonswould only form about 3% of the total mass of the fusion protein. Also,separate isolation or purification procedures (e.g., affinitypurification procedures) are generally required for each type of peptidereleased from a fusion protein. Release of the peptide from the fusionprotein generally involves use of specific chemical or enzymaticcleavage sites that link the carrier protein to the desired peptide[Forsberg et al., Int. J. Protein Chem., 11:201 (1992)]. Chemical orenzymatic cleavage agents employed for such cleavages generallyrecognize a specific sequence. However, if that cleavage sequence ispresent in the peptide of interest, then a different cleavage agent mustusually be employed. Use of a complex fusion partner (e.g.,β-galactosidase) that may have many cleavage sites produces a complexmixture of products and complicates isolation and purification of thepeptide of interest.

Chemical cleavage reagents in general recognize single or paired aminoacid residues that may occur at multiple sites along the primarysequence, and therefore may be of limited utility for release of largepeptides or protein domains which contain multiple internal recognitionsites. However, recognition sites for chemical cleavage can be useful atthe junction of short peptides and carrier proteins. Chemical cleavagereagents include cyanogen bromide, which cleaves at methionine residues[Piers et al., Gene, 134:7, (1993)], N-chloro succinimide [Forsberg etal., Biofactors, 2:105 (1989)] or BNPS-skatole [Knott et al., Eur. J.Biochem., 174:405 (1988); Dykes et al., Eur. J. Biochem., 174:411(1988)] which cleave at tryptophan residues, dilute acid which cleavesaspartyl-prolyl bonds [Gram et al., Bio/Technology, 12:1017 (1994);Marcus, Int. J. Peptide Protein Res., 25:542 (1985)], and hydroxylaminewhich cleaves asparagine-glycine bonds at pH 9.0 [Moks et al.,Bio/Technology, 5:379 (1987)].

For example, Shen describes bacterial expression of a fusion proteinencoding pro-insulin and β-galactosidase within insoluble inclusionbodies where the inclusion bodies were first isolated and thensolubilized with formic acid prior to cleavage with cyanogen bromide.Shen, Proc. Nat'l. Acad. Sci. (USA), 281:4627 (1984). Dykes et al.describes soluble intracellular expression of a fusion protein encodingα-human atrial natriuretic peptide and chloramphenicol acetyltransferasein E. coli where the fusion protein was chemically cleaved with2-(2-nitrophenylsulphenyl)-methyl-3′-bromoindolenine to release peptide.Dykes et al., Eur. J. Biochem., 174:411 (1988). Ray et al. describessoluble intracellular expression in E. coli of a fusion protein encodingsalmon calcitonin and glutathione-S-transferase where the fusion proteinwas cleaved with cyanogen bromide. Ray et al., Bio/Technology, 11:64(1993)

Proteases can provide gentler cleavage conditions and sometimes evengreater cleavage specificity than chemical cleavage reagents because aprotease will often cleave a specific site defined by the flanking aminoacids and the protease can often perform the cleavage underphysiological conditions. For example, Schellenberger et al. describesexpression of a fusion protein encoding a substance P peptide (11 aminoacids) and β-galactosidase within insoluble inclusion bodies, where theinclusion bodies were first isolated and then treated with chymotrypsinto cleave the fusion protein. Schellenberger et al., Int. J. PeptideProtein Res., 41:326 (1993). Pilon et al. describe soluble intracellularexpression in E. coli of a fusion protein encoding a peptide andubiquitin where the fusion protein was cleaved with a ubiquitin specificprotease, UCH-L3. Pilon et al., Biotechnol. Prog., 13:374 (1997). U.S.Pat. No. 5,595,887 to Coolidge et al. discloses generalized methods ofcloning and isolating peptides. U.S. Pat. No. 5,707,826 to Wagner et al.describes an enzymatic method for modification of recombinantpolypeptides.

Glucagon Like Peptide or GLP is an example of a polypeptide that can beproduced by recombinant methods. GLP-1 and GLP-2 are produced in vivo bycleavage of preproglucagon to produce the two bioactive polypeptides.The original sequencing studies indicated that GLP-2 includedthirty-four amino acids.

The recombinant production of any of these GLP peptides in high yield,however, is elusive because post expression manipulation usingtraditional methods provides poor results. Consequently, the goal ofrecombinant production of GLP through a one pot, high yield processlends itself to protease post-expression manipulation. Currentlyavailable processes cleavage of possible pre-GLP polypeptide substratesnecessitate use of different proteases and unique conditions and/orpre-or post-manipulation of the precursor polypeptides. Hence, improvedand simplified methods for making GLP peptides are needed. Inparticular, a simplified, high yield method for making GLP peptides isneeded.

SUMMARY OF THE INVENTION

These and other needs are achieved by the present invention which isdirected to a site specific clostripain cleavage of single and multicopypolypeptides having or containing a peptide sequence of the formulaGLP-2(1-33), GLP-2(1-33,A2G), GLP-2 (1-34) GLP-2 (1-34,A2G) andmutations, permutations and conservative substitutions thereof(hereinafter these peptides are termed the GLP-2 peptides as a group).In particular, the present invention is directed to a method thatsurprisingly selects a particular clostripain cleavage site from amongseveral that may be present in a single or multicopy polypeptide. Theresult of this surprising characteristic of the method of the inventionis the development of a versatile procedure for wide-ranging productionof desired polypeptides from single and multicopy polypeptides.

An especially preferred method according to the invention involves theproduction of any desired peptide through recombinant techniques. Thisfeature is accomplished through use of a single copy polypeptide havinga discardable sequence ending in arginine joined to the N-terminus ofthe desired peptide. The cleavage of that designated arginine accordingto the invention is so selective that the desired peptide may containany sequence of amino acids. The cleavage produces a single copy of thedesired peptide. Thus, the methods according to the invention enable theproduction of polypeptides having C-terminal acidic, aliphatic oraromatic amino acid residues and the production of a GLP-2(1-33) orGLP-2(1-34) peptide. Some of the salient details of these methods of theinvention are summarized in the following passages.

The invention provides methods for making peptides using clostripaincleavage of a larger polypeptide that has at least one copy of any ofthe GLP-2 peptides. According to the invention, clostripain recognizes apolypeptide having a site as indicated in Formula I and cleaves apeptide bond between amino acids Xaa₂ and Xaa₃:Xaa₁-Xaa₂-Xaa₃  Formula Iwherein Xaa₁ and Xaa₃ may be any non-acidic amino acid residue and Xaa₂is arginine. According to a preferable aspect of the invention,clostripain selectively recognizes a the site as indicated in Formula Iand cleaves the peptide bond between amino acids Xaa₂ and Xaa₃ whereinXaa₁ is an amino acid residue with an acidic side chain such as asparticacid, or glutamic acid, or non-acidic amino acid such as proline orglycine; Xaa₂ is arginine; and Xaa₃ is not an acidic amino acid. Also,through the control of any one or more of pH, time, temperature andreaction solvent involved in the cleavage reaction, the rate andselectivity of the clostripain cleavage may be manipulated. Thus, forexample, the GLP-2(1-34) peptide of the sequence

HADGSFSDGMNTILDNLAARDFINWLIQTKITDR SEQ ID NO: 9may be formed as multiple copies coupled together with a linker of anappropriate sequence, or multiple copies coupled together in tandem withthe N-terminus (H) forming a peptide bond with the C-terminus (R) of theupstream copy, or a discardable sequence ending with Xaa₁-Xaa₂-Xaa₃coupled to the N-terminus, or beginning with Xaa₃ coupled to theC-terminus, of the desired peptide.

Clostripain will eventually cleave the peptide bond on the carboxyl sideof any arginine or lysine appearing in an amino acid sequenceirrespective of the amino acid residues adjacent arginine. Surprisingly,it has been discovered that the rate of clostripain cleavage of apolypeptide can be dramatically altered by specifically altering aminoacids immediately on the N-terminal and C-terminal side of an arginineresidue that acts as a clostripain cleavage site. In particular,according to the invention, this preferred clostripain cleavage of anarginine amino acid residue peptide bond can be manipulated to be highlyselective through use of an acidic amino acid residue bonded to theamine side of arginine, eg. Xaa₁ of foregoing Formula I. According tothe invention, it has also been discovered that by manipulation of anyone or more of pH, time, temperature and solvent character, the rate ofclostripain cleavage can be manipulated to affect cleavage of a selectedXaa₂-Xaa₃ peptide bond of Formula I. Combinations of these factors willenable selection of particular arginine—amino acid residue bonds fromamong several differing such bonds that may be present in the precursorpolypeptide.

In one aspect, the invention provides a method for producing a desiredpeptide from a polypeptide by cleaving at least one peptide bond withinthe polypeptide using clostripain. The clostripain cleaves a peptidebond between amino acids Xaa₂ and Xaa₃ of a polypeptide having theFormula II:(Xaa₃-Peptide₁-Xaa₁-Xaa₂)_(n)-Xaa₃-Peptide₁-Xaa₁-Xaa₂  Formula IIIn this aspect of the invention, the desired GLP-2 peptides have theFormula Xaa₃-Peptide₁-Xaa₁-Xaa₂. Also in this aspect of the invention, nis an integer ranging from 0 to 50. Xaa₁ is aspartic acid, glycine,proline or glutamic acid. Xaa₂ is arginine. Xaa₃ is not an acidic aminoacid.

In another aspect, the invention provides a method for producing adesired peptide, such as GLP-2(1-33) or GLP-2 (1-34). Such a methodinvolves cleaving with clostripain a peptide bond between amino acidsXaa₂ and Xaa₃ within a polypeptide comprising Formula III:(Linker-Xaa₃-Peptide₁)_(n)-Linker-Xaa₃-Peptide₁  Formula IIIIn this aspect of the invention, the desired peptide GLP-2 has theFormula Xaa₃-Peptide₁. n is an integer ranging from 0 to 50. Xaa₃ is notan acidic amino acid. Linker is a cleavable peptide linker havingFormula IV:(Peptide₅)_(m)-Xaa₁-Xaa₂  Formula IVm is an integer ranging from 0 to 50. Xaa₁ is aspartic acid, glycine,proline or glutamic acid. Xaa₂ is arginine. Peptide₅ is any single ormulti amino acid sequence not containing the sequence Xaa₁-Xaa₂.

The invention further provides a method of producing a GLP-2(1-34)peptide. The method involves the steps of

-   -   (a) recombinantly producing a polypeptide of the Formula VI:        Tag-Linker-[GLP-2(1-34)]_(q)  VI        -   wherein Tag is a translation initiation sequence having SEQ            ID NO:17 or 18; Linker is a cleavable peptide linker of            Formula IV described above; GLP-2(1-34) has SEQ ID NO:9; and            q is an integer of about 2 to about 20;    -   (b) isolating the polypeptide of Formula VI; and    -   (c) cleaving at least one peptide bond within the polypeptide of        Formula VI using clostripain, wherein clostripain cleaves a        peptide bond on the C-terminal side of Xaa₂.

The invention also provides a method of producing a GLP-2 peptide frominclusion bodies. The method involves the steps of:

-   -   (a) recombinantly producing a polypeptide of the Formula V        within inclusion bodies of a bacterial host cell:        Tag-IBFP-Linker-GLP-2  V        -   wherein:            -   Tag is a translation initiation sequence comprising SEQ                ID NO:17 or 18;            -   IBFP is an inclusion body leader partner comprising any                one of SEQ ID NO:19, 20, 21 or 22;            -   Linker is a cleavable peptide linker having Formula IV:                (Peptide₅)_(m)-Xaa₁-Xaa₂  IV        -   wherein:            -   n is an integer ranging from 0 to 50;            -   m is an integer ranging from 0 to 50;            -   Xaa₁ is aspartic acid, glycine, proline, or glutamic                acid;            -   Xaa₂ is arginine; and            -   Peptide_(s) is a single amino acid residue or a multiple                amino acid sequence; and        -   GLP-2 has any of the sequences given for the GLP-2 peptides;    -   (b) isolating the bacterial inclusion bodies;    -   (c) solubilizing the inclusion bodies containing the polypeptide        of Formula V using urea;    -   (d) cleaving, in the presence of about 0 M to about 8 M urea, at        least one peptide bond within the polypeptide of Formula V using        clostripain, wherein clostripain cleaves a peptide bond on the        C-terminal side of Xaa₂.

The invention also includes methods of transpeptidation and C-terminusamidation. In particular, the invention also provides a method ofproducing a GLP-2 peptide amide or extension. The method involves thesteps of:

-   -   (a) recombinantly producing a polypeptide of the Formula VIII:        Tag-Linker-[GLP-2-Linker₂]_(q)  VIII        -   wherein:            -   Tag is an amino acid sequence comprising SEQ ID NO:17 or                18;            -   Linker is a cleavable peptide linker having Formula IV:                (Peptide₅)_(m)-Xaa₁-Xaa₂  IV        -   wherein:            -   n is an integer ranging from 0 to 50;            -   m is an integer ranging from 0 to 50;            -   Xaa₁ is aspartic acid, glycine, proline, or glutamic                acid;            -   Xaa₂ is arginine; and            -   Peptide₅ is any amino acid combination;        -   Linker₂ is SEQ ID NO:23;        -   GLP-2 is any of the GLP-2 peptide sequences described            herein;        -   q is an integer of about 2 to about 20;    -   (b) isolating the polypeptide of Formula VIII;        cleaving at least one peptide bond within the polypeptide of        Formula VIII using clostripain in the presence of ammonia,        wherein clostripain cleaves a peptide bond on the C-terminal        side of Xaa₂, amidates the carbonyl of Xaa₂ and thereby forms a        GLP-2(1-34)NH₂ peptide having SEQ ID NO: 10, or a GLP-2(1-33)NH₂        peptide having SEQ ID NO:12. Alternatively, glycine instead of        ammonia can be included within the clostripain cleavage to        produce a GLP-2(1-33) peptide.

Finally, additional aspects of the invention include modificationsregarding production of polypeptide within a bacterial cell. A DNAsegment encoding the precursor polypeptide can be transformed into thebacterial host cells. The DNA segment can also encode a peptidylsequence linked to the precursor polypeptide wherein the peptidylsequence encourages the polypeptide to be sequestered within bacterialinclusion bodies. Such peptidyl sequences are termed “inclusion bodyleader partners” and include peptidyl sequences having, for example, SEQID NO:19, 20, 21 or 22. Use of such an inclusion body leader partnerfacilitates isolation and purification of the polypeptide. Isolation ofthe bacterial inclusion bodies containing the polypeptide is simple(e.g., centrifugation). According to the invention, the isolatedinclusion bodies can be used without substantial purification, forexample, by solubilizing the polypeptide in urea and then conducting theclostripain cleavage reaction either before or after removal of theurea. Clostripain is capable of cleaving polypeptide in comparativelyhigh concentrations of urea, for example, in the presence of about 0 Mto about 8 M urea, so removal of urea is not required. Hence, theinvention provides methods for cleaving a soluble polypeptide, or aninsoluble polypeptide that can be made soluble by adding urea.

The invention also includes an assay for measuring the development ofinclusion bodies. The method involves use of a phenolic medium andseparation/analytic techniques. Details are given within the Examplessection.

DESCRIPTION OF THE DRAWINGS

FIG. 1 provides schematic diagrams of A: a pBN121 vector containing aDNA segment encoding the precursor polypeptide, T7tag-GSDR(GLP-2(1-34)₆(SEQ ID NO:37). B: a pBN122 vector containing a DNA segment encoding theprecursor polypeptide. T7tagVg-VDDR-GLP-2(1-33,A2G) PYX (SEQ ID NO:38);chlorella Virus Promoter.

FIG. 2 illustrates a typical growth curve of recombinant E. coli.Addition of IPTG generally occurs between 10 and 11 hours. Cells areharvested 6 to 10 hours after induction.

FIGS. 3A and 3B illustrate an LC-MS analysis of cell free extracts froma typical fermentation producing T7tagVg-GSDR-[GLP-2(1-34)]₆ (SEQ IDNO:37) (about 9 gm/L); FIGS. 3C and 3D illustrate HPLC analysis of cellfree extracts of C) T7tagVg-VDDR-GLP-2(1-33,A2G) (SEQ ID NO:40) (8.7gm/L); or D: T7tag-GSDR-GLP-1(1-33)A2G-PGDR-GLP-2(1-33,A2G) (SEQ IDNO:39) (10.4 gm/L). In each case cell samples were taken after 10 hoursof induction and prepared for analysis as described in the text.

FIG. 4 illustrates the digestion of a precursor polypeptide,T7tag-GSDR-[GLP-2(1-34)]₆ (SEQ ID NO:37) in a cell free extract withclostripain to produce GLP-2(1-34)(closed squares) and a GLP-2 fragment(21-34)(closed circle). The digestion was conducted by combining 0.1unit of clostripain per mg of the precursor polypeptide. The precursorpolypeptide was present in the digestion mixture at a concentration ofabout 0.45 mg/ml. FIG. 4A illustrates the digestion of a precursorpolypeptide, T7tagVg-VDDR-GLP-2(1-33,A2G) (SEQ ID NO:40), in a cell freeextract with clostripain to produce GLP-2(1-33, A2G) and a GLP-2fragment (21-33). The digestion was conducted by combining 0.2 units ofclostripain per mg of the precursor polypeptide. The precursorpolypeptide was present in the digestion mixture at a concentration ofabout 0.45 mg/ml (closed triangle) T7tagVg-VDDR-GLP-2(1-33,A2G) (SEQ IDNO:40); (closed diamond) GLP-2(1-33,A2G); (closed circle) GLP-2 fragment(21-33).

FIG. 5 shows the results of a liquid chromatography-mass spectroscopy(LC/MS) conducted on the products of the precursor polypeptide,T7tag-GSDR-[GLP-2(1-34)]₆ (SEQ ID NO:37), after digestion withclostripain. The mass spectra represent the masses of the peak fromHPLC. Mass spectrum A was from peak at 8.25 minutes, B from peak at 8.6minutes, C from peak at 10.24 minutes and D from peak at 11.2 minutes.Peak A was identified as GLP-2(1-20) and had a mass of 2177; Peak B wasidentified as GLP-2 (21-34) and had a mass of 1763, Peak C wasidentified as GLP-2(1-34) and had a mass of 3922; and peak D wasidentified as [(GLP-2(1-34)]₂ and has a mass of 7826.

FIG. 5A illustrates typical liquid chromatography-mass spectroscopy(LC-MS) analysis of the reaction products of a clostripain digestion ofa precursor polypeptide. Panel (A) shows the relative abundancechromatogram. Panel (B) shows the absorbance chromatogram at A280 nm.Panel (C) shows the mass of peak 1 of panel (A) which correlated toGLP-2(21-33). Panel (D) shows the mass of peak 2 of panel (A) whichcorresponds to GLP-2(1-33,A2G)-PGDR (SEQ ID NO:41). Panel (E) shows themass of peak 3 in panel (A) which corresponds to GLP-2(1-33,A2G).

FIG. 6 shows a plot of peak area of GLP-2(1-34) produced from thecleavage reaction as a function of time under different pH conditions;(closed triangles) pH 6.0; (closed diamonds) pH 6.5; (closed squares) pH7.05; (+ signs) pH 7.63; (open squares) pH 8.0.

FIG. 6A: illustrates clostripain digestion under different pHconditions. peak 1: GLP-2(21-33), peak 2: T7Vg-GLP-2(1-33,A2G), peak 3:AMVDDR-GLP-2(1-33,A2G) (SEQ ID NO:42), peak 4:GSGQGQAQYLAASLVVFTNYSGDTASQVDVVGPRAMVDDR-GLP-2(1-33,A2G) (SEQ ID NO:43),and peak 5: GLP-2(1-33,A2G). At pH 6.9, the greatest transformation ofthe precursor polypeptide to the peptide product was achieved. Theclostripain cleavage reaction above pH 7 is less specific; FIG. 6B:shows a plot of percent yield of GLP-2(1-33,A2G) produced from thecleavage reaction as a function of time under different ureaconcentrations (closed circle) 0 M; (closed diamond) 0.5 M; (closedsquare) 1.0 M; (closed triangle) 1.5 M urea.

FIG. 7A shows the effect of precursor polypeptide concentration on therate of cleavage by clostripain. FIG. 7B shows the effect of clostripainconcentration on the rate of cleavage of a precursor polypeptide.

FIG. 8 shows purified GLP-2 (1-34) obtained through use of the methodsdescribed in Example 8. Panel A illustrates an HPLC analysis of thepurified peptide at the retention time of 26.9 minutes. Panel Billustrates the mass spectrum of the peptide, in which the 1308.5 m/zvalue is the 3+ charged species. Accordingly the mass is 3922.5 whichconfirms the identity of GLP-2(1-34).

FIGS. 9A and 9B illustrate the effect of organic solvents on the rateand extent of cleavage of a precursor polypeptideT7tagVg-VDDR-GLP-2(1-33, A2G) by clostripain. FIG. 9A: (dashed line is10% ethanol) (dotted line is 20% ethanol) (solid line is 35% ethanol).Peak 1 is GLP-2(21-33), Peak 2 is GLP-2(1-33,A2G) (SEQ ID NO:40) andPeak 3 is the T7tagVg-VDDR-GLP-2(1-33,A2G) (SEQ ID NO:40) precursorpolypeptide. FIG. 9B: (closed square) rate of formation ofGLP-2(1-33,A2G) in 30% ethanol; (closed triangle) rate of formation ofGLP-2(1-33,A2G) in 30% acetonitrile; (closed circle) rate of formationof GLP-2(1-33,A2G) in the absence of organic solvent; (open circle) rateof formation of GLP-2(21-33) in the absence of an organic solvent; (opentriangle) rate of formation of GLP-2(21-33) in 30% acetonitrile; (opensquare) rate of formation of GLP-2(21-33) in 30% ethanol.

FIG. 10 illustrates is an analytical reverse phase HPLC of purifiedGLP-2(1-33,A2G). Chromatography was performed using an Alltima C18column (7×33 mm, resin size is 3.5 μm). The mobile phase was (A) 0.1%TFA in water and (B) 0.08% TFA in acetonitrile. The flow rate was 2 mlper minute and the column was maintained at 50° C. The column wasequilibrated with 15% (B) and 85% (A). The gradient was formed from 15%to 45% (B) in 1 minute, 35% to 50% (B) in 5 minutes, and 50% to 90% (B)in 1 minute. The GLP-2(1-33,A2G) eluted in about 4.8 minutes.

DEFINITIONS OF THE INVENTION

Abbreviations: LC-MS: liquid chromatography-mass spectroscopy; TFA:trifloroacetic acid; DTT: dithiothreitol; DTE: dithioerythritol.

An “Amino acid analog” includes amino acids that are in the D ratherthan L form, as well as other well-known amino acid analogs, e.g.,N-alkyl amino acids, lactic acid, and the like. These analogs includephosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline,gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylicacid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,penicillamine, ornithine, citruline, N-methyl-alanine,para-benzoyl-phenylalanine, phenylglycine, propargylglycine, sarcosine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,norleucine, norvaline, orthonitrophenylglycine and other similar aminoacids.

The terms, “cells,” “cell cultures”, “Recombinant host cells”, “hostcells”, and other such terms denote, for example, microorganisms, insectcells, and mammalian cells, that can be, or have been, used asrecipients for nucleic acid constructs or expression cassettes, andinclude the progeny of the original cell which has been transformed. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement as the original parent, due to natural, accidental, ordeliberate mutation. Many cells are available from ATCC and commercialsources. Many mammalian cell lines are known in the art and include, butare not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), and humanhepatocellular carcinoma cells (e.g., Hep G2). Many prokaryotic cellsare known in the art and include, but are not limited to, Escherichiacoli and Salmonella typhimurium. Sambrook and Russell, MolecularCloning: A Laboratory Manual, 3rd edition (Jan. 15, 2001) Cold SpringHarbor Laboratory Press, ISBN: 0879695765. Many insect cells are knownin the art and include, but are not limited to, silkworm cells andmosquito cells. (Franke and Hruby, J. Gen. Virol., 66:2761 (1985);Marumoto et al., J. Gen. Virol., 68:2599 (1987)).

A “cleavable peptide linker” refers to a peptide sequence having aclostripain cleavage recognition sequence.

A “coding sequence” is a nucleic acid sequence that is translated into apolypeptide, such as a preselected polypeptide, usually via mRNA. Theboundaries of the coding sequence are determined by a translation startcodon at the 5′-terminus and a translation stop codon at the 3′-terminusof an mRNA. A coding sequence can include, but is not limited to, cDNA,and recombinant nucleic acid sequences.

A “conservative amino acid” refers to an amino acid that is functionallysimilar to a second amino acid. Such amino acids may be substituted foreach other in a polypeptide with minimal disturbance to the structure orfunction of the polypeptide. The following five groups each containamino acids that are conservative substitutions for one another:Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine(I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W);Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine (R),Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E),Neutral: Asparagine (N), Glutamine (Q). Examples of other synthetic andnon-genetically encoded amino acid types are provided herein.

The term “gene” is used broadly to refer to any segment of nucleic acidthat encodes a preselected polypeptide. Thus, a gene may include acoding sequence for a preselected polypeptide and/or the regulatorysequences required for expression. A gene can be obtained from a varietyof sources. For example, a gene can be cloned or PCR amplified from asource of interest, or it can be synthesized from known or predictedsequence information.

An “inclusion body” is an amorphous polypeptide deposit in the cytoplasmof a cell. In general, inclusion bodies comprise aggregated protein thatis improperly folded or inappropriately processed.

An “inclusion body leader partner” is a peptide that causes apolypeptide to which it is attached to form an inclusion body whenexpressed within a bacterial cell. The inclusion body leader partners ofthe invention can be altered to confer isolation enhancement onto aninclusion body that contains the altered inclusion body leader partner.

The term “lysate” as used herein refers to the product resulting fromthe breakage of cells. Such cells include both prokaryotic andeukaryotic cells. For example, bacteria may be lysed though a largenumber of art recognized methods. Such methods include, but are notlimited to, treatment of cells with lysozyme, French press, treatmentwith urea, organic acids, and freeze thaw methods. Methods for lysingcells are known and have been described. (Sambrook and Russell,Molecular Cloning: A Laboratory Manual, 3rd edition (Jan. 15, 2001) ColdSpring Harbor Laboratory Press, ISBN: 0879695765; Stratagene, La Jolla,Calif.).

An “open reading frame” (ORF) is a region of a nucleic acid sequencethat encodes a polypeptide.

“Operably-linked” refers to the association of nucleic acid sequences oramino acid sequences on a single nucleic acid fragment or a single aminoacid sequence so that the function of one is affected by the other. Forexample, a regulatory DNA sequence is said to be “operably linked to” or“associated with” a DNA sequence that codes for an RNA if the twosequences are situated such that the regulatory DNA sequence affectsexpression of the coding DNA sequence (i.e., that the coding sequence orfunctional RNA is under the transcriptional control of the promoter). Inan example related to amino acid sequences, an inclusion body leaderpartner is said to be operably linked to a preselected amino acidsequence when the inclusion body leader partner causes a precursorpolypeptide to form an inclusion body. In another example, a signalsequence is said to be operably linked to a preselected amino acid whenthe signal sequence directs the precursor polypeptide to a specificlocation in a cell.

The term “polypeptide” refers to a polymer of amino acids and does notlimit the size to a specific length of the product. However, as usedherein, a polypeptide is generally longer than a peptide and may includeone or more copies of a peptide of interest (the terms peptide ofinterest and desired peptide are used synonymously herein). This termalso optionally includes post expression modifications of thepolypeptide, for example, glycosylations, acetylations, phosphorylationsand the like. Included within the definition are, for example,polypeptides containing one or more analogues of an amino acid orlabeled amino acids.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter” includes a minimal promoter that isa short DNA sequence comprised of a TATA-box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. “Promoter” alsorefers to a nucleotide sequence that includes a minimal promoter plusregulatory elements that is capable of controlling the expression of acoding sequence. Promoters may be derived in their entirety from anative gene, or be composed of different elements derived from differentpromoters found in nature, or even be comprised of synthetic DNAsegments. A promoter may also contain DNA segments that are involved inthe binding of protein factors that control the effectiveness oftranscription initiation in response to physiological or environmentalconditions.

The term “purification stability” refers to the isolationcharacteristics of an inclusion body formed from a polypeptide having aninclusion body leader partner operably linked to a polypeptide. Highpurification stability indicates that an inclusion body can be isolatedfrom a cell in which it was produced. Low purification stabilityindicates that the inclusion body is unstable during purification due todissociation of the polypeptides forming the inclusion body.

When referring to a polypeptide or nucleic acid, “isolated” means thatthe polypeptide or nucleic acid has been removed from its naturalsource. An isolated polypeptide or nucleic acid may be present within anon-native host cell and so the polypeptide or nucleic acid is thereforenot necessarily “purified.”

The term “purified” as used herein preferably means at least 75% byweight, more preferably at least 85% by weight, more preferably still atleast 95% by weight, and most preferably at least 98% by weight, ofbiological macromolecules of the same type present (but water, buffers,and other small molecules, especially molecules having a molecularweight of less than 1000, can be present).

“Regulated promoter” refers to a promoter that directs gene expressionin a controlled manner rather than in a constitutive manner. Regulatedpromoters include inducible promoters and repressable promoters. Suchpromoters may include natural and synthetic sequences as well assequences that may be a combination of synthetic and natural sequences.Different promoters may direct the expression of a gene in response todifferent environmental conditions. Typical regulated promoters usefulin the invention include, but are not limited to, promoters used toregulate metabolism (e.g., an IPTG-inducible lac promoter) heat-shockpromoters (e.g., an SOS promoter), and bacteriophage promoters (e.g., aT7 promoter).

A “ribosome-binding site” is a DNA sequence that encodes a site on anmRNA at which the small and large subunits of a ribosome associate toform an intact ribosome and initiate translation of the mRNA. Ribosomebinding site consensus sequences include AGGA or GAGG and are usuallylocated some 8 to 13 nucleotides upstream (5′) of the initiator AUGcodon on the mRNA. Many ribosome-binding sites are known in the art.(Shine et al., Nature, 254:34, (1975); Steitz et al., “Genetic signalsand nucleotide sequences in messenger RNA”, in: Biological Regulationand Development: Gene Expression (ed. R. F. Goldberger) (1979)).

A “selectable marker” is generally encoded on the nucleic acid beingintroduced into the recipient cell. However, co-transfection ofselectable marker can also be used during introduction of nucleic acidinto a host cell. Selectable markers that can be expressed in therecipient host cell may include, but are not limited to, genes whichrender the recipient host cell resistant to drugs such as actinomycinC₁, actinomycin D, amphotericin, ampicillin, bleomycin, carbenicillin,chloramphenicol, geneticin, gentamycin, hygromycin B, kanamycinmonosulfate, methotrexate, mitomycin C, neomycin B sulfate, novobiocinsodium salt, penicillin G sodium salt, puromycin dihydrochloride,rifampicin, streptomycin sulfate, tetracycline hydrochloride, anderythromycin. (Davies et al., Ann. Rev. Microbiol., 32:469, (1978)).Selectable markers may also include biosynthetic genes, such as those inthe histidine, tryptophan, and leucine biosynthetic pathways. Upontransfection or transformation of a host cell, the cell is placed intocontact with an appropriate selection marker.

The term “self-adhesion” refers to the association between polypeptidesthat have an inclusion body leader partner to form an inclusion body.Self-adhesion may affect the purification stability of an inclusion bodyformed from the polypeptide. Self-adhesion that is too great producesinclusion bodies having polypeptides that are so tightly associated witheach other that it is difficult to separate individual polypeptides froman isolated inclusion body. Self-adhesion that is too low producesinclusion bodies that are unstable during isolation due to dissociationof the polypeptides that form the inclusion body. Self-adhesion can beregulated by altering the amino acid sequence of an inclusion bodyleader partner.

A “signal sequence” is a region in a protein or polypeptide responsiblefor directing an operably linked polypeptide to a cellular location orcompartment designated by the signal sequence. For example, signalsequences direct operably linked polypeptides to the inner membrane,periplasmic space, and outer membrane in bacteria. The nucleic acid andamino acid sequences of such signal sequences are well-known in the artand have been reported. Watson, Molecular Biology of the Gene, 4thedition, Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif.(1987); Masui et al., in: Experimental Manipulation of Gene Expression,(1983); Ghrayeb et al., EMBO J., 3:2437 (1984); Oka et al., Proc. Natl.Acad. Sci. USA, 82:7212 (1985); Palva et al., Proc. Natl. Acad. Sci.USA, 79:5582 (1982); U.S. Pat. No. 4,336,336).

Signal sequences, preferably for use in insect cells, can be derivedfrom genes for secreted insect or baculovirus proteins, such as thebaculovirus polyhedrin gene (Carbonell et al., Gene, 73:409 (1988)).Alternatively, since the signals for mammalian cell posttranslationalmodifications (such as signal peptide cleavage, proteolytic cleavage,and phosphorylation) appear to be recognized by insect cells, and thesignals required for secretion and nuclear accumulation also appear tobe conserved between the invertebrate cells and vertebrate cells, signalsequences of non-insect origin, such as those derived from genesencoding human α-interferon (Maeda et al., Nature, 315:592 (1985)),human gastrin-releasing peptide (Lebacq-Verheyden et al., Mol. Cell.Biol., 8:3129 (1988)), human IL-2 (Smith et al., Proc. Natl. Acad. Sci.USA, 82:8404 (1985)), mouse IL-3 (Miyajima et al., Gene, 58:273 (1987))and human glucocerebrosidase (Martin et al., DNA, 7:99 (1988)), can alsobe used to provide for secretion in insects.

The term “solubility” refers to the amount of a substance that can bedissolved in a unit volume of solvent. For example, solubility as usedherein refers to the ability of a polypeptide to be resuspended in avolume of solvent, such as a biological buffer.

A “Tag” sequence refers to an amino acid sequence that is operablylinked to the N-terminus of a peptide or protein. Such tag sequences mayprovide for the increased expression of a desired peptide or protein.Such tag sequences may also form a cleavable peptide linker when theyare operably linked to another peptide or protein. Examples of tagsequences include, but are not limited to, the sequences indicated inSEQ ID NOs: 17 and 18.

A “transcription terminator sequence” is a signal within DNA thatfunctions to stop RNA synthesis at a specific point along the DNAtemplate. A transcription terminator may be either rho factor dependentor independent. An example of a transcription terminator sequence is theT7 terminator. Transcription terminators are known in the art and may beisolated from commercially available vectors according to recombinantmethods known in the art. (Sambrook and Russell, Molecular Cloning: ALaboratory Manual, 3rd edition (Jan. 15, 2001) Cold Spring HarborLaboratory Press, ISBN: 0879695765; Stratagene, La Jolla, Calif.).

“Transformation” refers to the insertion of an exogenous nucleic acidsequence into a host cell, irrespective of the method used for theinsertion. For example, direct uptake, transduction, f-mating orelectroporation may be used to introduce a nucleic acid sequence into ahost cell. The exogenous nucleic acid sequence may be maintained as anon-integrated vector, for example, a plasmid, or alternatively, may beintegrated into the host genome.

A “translation initiation sequence” refers to a DNA sequence that codesfor a sequence in a transcribed mRNA that is optimized for high levelsof translation initiation. Numerous translation initiation sequences areknown in the art. These sequences are sometimes referred to as leadersequences. A translation initiation sequence may include an optimizedribosome-binding site. In the present invention, bacterial translationalstart sequences are preferred. Such translation initiation sequences areavailable in the art and may be obtained from gene 10 of bacteriophageT7, and the gene encoding ompT. Those of skill in the art can readilyobtain and clone translation initiation sequences from a variety ofcommercially available plasmids, such as the pET series of plasmids.(Stratagene, La Jolla, Calif.).

A “unit” of clostripain activity is defined as the amount of enzymerequired to transform 1 μmole of benzoyl-L-arginine ethyl ester (BAEE)to benzoyl-L-arginine per minute at 25° C. under defined reactionconditions. The transformation is measured spectroscopically at 253 nm.The assay solution contained 2.5 mM BAEE, 10 mM HEPES (pH 6.7), 2 mMCaCl₂, and 1 mM DTT.

A “variant” polypeptide is intended a polypeptide derived from thereference polypeptide by deletion, substitution or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativepolypeptide; deletion or addition of one or more amino acids at one ormore sites in the native protein; or substitution of one or more aminoacids at one or more sites in the reference protein. Such substitutionsor insertions are preferably conservative amino acid substitutions.Methods for such manipulations are generally known in the art. Kunkel,Proc. Natl. Acad. Sci. USA, 82:488, (1985); Kunkel et al., Methods inEnzymol., 154:367 (1987); U.S. Pat. No. 4,873,192; Walker and Gaastra,eds. (1983) Techniques in Molecular Biology (MacMillan PublishingCompany, New York) and the references cited therein. Guidance as toappropriate amino acid substitutions that do not affect biologicalactivity of the protein of interest may be found in the model of Dayhoffet al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed.Res. Found., Washington, D.C.).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for efficiently making peptides of theformulas GLP-2(1-33), GLP-2(1-33, A2G), GLP-2(1-34), GLP-2(1-34,A2G) andmutations, permutations and conservative substitutions thereof(hereinafter these peptides are termed the GLP-2 peptides as a group).The peptides are made using recombinant and proteolytic procedures. Theinvention enables the wide-ranging use of a single cleavage enzyme whoseselectivity can be manipulated. In particular, the enzyme, clostripain,can be manipulated to cleave a particular site when the same primarycleavage site appears elsewhere in the peptide. Although limited toinitial cleavage at a C-terminal side of arginine residues, the methodprovides versatility. The versatility arises from the surprising abilityto manipulate clostripain so that it will cleave at the C-terminus eventhough arginine or lysine appears elsewhere within the peptide sequence.

The need to avoid reassimilation of an expressed, desired peptide byhost expression cells dictates that the desired peptide should have asignificantly high molecular weight and varied amino acid sequence. Suchpeptide features are desirable when recombinant peptides are beingproduced. This need means that the expressed polypeptide be formedeither as a multicopy of the desired peptide or as a combination of thedesired peptide be linked to a discardable peptide sequence. Use of theformer multicopy scheme provides multiple copies of the desired peptideunder certain circumstances and the desired peptide with severaladditional amino acid residues at its N- and C-termini under all othercircumstances. Use of the latter single copy scheme provides at least asingle copy of the desired peptide.

According to the invention, the latter scheme may be employed to producevirtually any desired peptide. The discardable sequence is manipulatedaccording to the invention in part to have arginine as its carboxyl end.The arginine is in turn coupled by its peptide bond to the N-terminus ofthe desired peptide. The cleavage of that designated arginine accordingto the invention is so selective that the desired peptide may containvirtually any sequence of amino acids. The cleavage produces a singlecopy of the desired peptide.

Although it is not to be regarded as a limitation of the invention, theselectivity of this enzymatic cleavage is believed to be the result ofthe influence of secondary binding sites of the substrate with theenzyme, clostripain. These secondary sites are adjacent to the primarycleavage site and are known as the P and P′ sites. There may be one ormultiple P and P′ sites. The P sites align with the amino acid residueson the amino side of the scissile bond while the P′ sites align with theamino acid residues on the carboxyl side of the scissile bond. Thus, thescissile bond resides between the P and the P′ bond. The correspondingsites of the enzyme are termed S and S′ sites. It is believed that theside chain character of the P and P′ amino acid residues immediatelyadjacent the primary cleavage residue have significant influence uponthe ability of the enzyme to bind with and cleave the peptide bond atthe primary cleavage site.

For clostripain, it has been discovered that an acidic amino acidresidue occupying the P₂ site (amino side) immediately adjacent to theP₁ primary cleavage amino acid residue, arginine, causes highlyselective, rapid attack of clostripain upon that particular primarycleavage site. It has also been discovered that an acidic amino acidresidue occupying the P₁′ site (carboxyl side) immediately adjacent theprimary cleavage site causes repulsion of, and extremely slow attack of,clostripain upon the primary cleavage site.

Thus, according to a preferred method of the invention, a polypeptidethat has at least one copy of a peptide of interest may be recombinantlyproduced. The production may be of a soluble polypeptide or an inclusionbody preparation containing at least a substantially insoluble mass ofpolypeptide. Next, the polypeptide is proteolytically cleaved usingclostripain to produce the peptide of interest. By manipulating thepolypeptide and/or the cleavage conditions, peptides having anyC-terminal residue can be produced. Further, by using the method of thisinvention, peptides having any C-terminal residue amide can be produced.For example GLP-2(1-33)NH₂ or GLP-2(1-34)NH₂ can be produced fromGLP-2(1-33)CH or GLP-2(1-34)CH respectively through use of the method ofthis invention.

The Clostripain Cleavage Process According to the Invention

According to the invention, clostripain is used in a selective manner toaffect preferential cleavage at a selected arginine site. As explainedbelow, clostripain is recognized to cleave at the carboxyl side ofarginine and lysine residues in peptides. One of the surprising featuresof the present invention is the discovery of the ability to provide aselective cleavage site for clostripain so that it will preferentiallycleave at a designated arginine even though other arginine or lysineresidues are present within the peptide. Multicopy polypeptides havingarginine residues at the inchoate C-termini of the desired peptideproduct copies within the polypeptide and also having arginine or lysineresidues within the desired peptide sequence can be efficiently andselectively cleaved according to the invention to produce the desiredpeptide product.

Moreover, the enzymatic cleavage, precursor polypeptide and desiredpeptide product can be manipulated so that the C-terminus of the peptideproduct may be any amino acid residue. This feature is surprising inview of the cleavage preference of clostripain toward arginine. Thisfeature is accomplished through use of a discardable sequence ending inarginine and joined to the N-terminus of the desired peptide. Thecleavage of that designated arginine according to the invention is soselective that the desired peptide may contain virtually any sequence ofamino acids. The cleavage produces a single copy of the desired peptide.

Traditional Clostripain Cleavage Conditions

Clostripain (EC 3.4.22.8) is an extracellular protease from Clostridiathat can be recovered from the culture filtrate of Clostridiumhistolyticum. Clostripain has both proteolytic and amidase/esteraseactivity. Mitchell et al., Biol. Chem., 243:4683 (1968). Clostripain isa heterodimer with a molecular weight of about 50,000 and an isoelectricpoint of pH 4.8 to 4.9. Clostripain proteolytic activity is inhibited,for example, by tosyl-L-lysine chloromethyl ketone, hydrogen peroxide,Co⁺⁺, Cu⁺⁺ or Ca⁺⁺ ions, citrate, or Ca⁺⁺ chelators, such as EGTA andEDTA. Examples of clostripain activators include cysteine,mercaptoethanol, dithiothreitol and calcium ions.

Clostripain is generally understood to have specificity for cleavage ofArg-Xaa linkages, though some cleavage can occur at lysine residuesunder certain reaction conditions. Thus, in the isolated B chain ofinsulin, clostripain cleaves the Arg-Gly linkage 500 times more rapidlythan the Lys-Ala linkage. In glucagon, only the Arg-Arg, the Arg-Ala andthe Lys-Tyr sites are cleaved. The relative initial rates of hydrolysisof these three bonds are 1, 1/7 and 1/300. (Labouesses, Bull. Soc. Chim.Biol., 42:1293, (1960)).

Clostripain Cleavage According to the Invention

According to the invention, amino acids flanking arginine can stronglyinfluence clostripain cleavage. In particular, clostripain has a strongpreference for a polypeptide having a cleavage site shown by Formula I,where the cleavage occurs at a peptide bond after amino acid Xaa₂:Xaa₁-Xaa₂-Xaa₃  (I)

-   -   wherein        -   Xaa₁ aspartic acid, glycine, proline or glutamic acid;        -   Xaa₂ is arginine; and        -   Xaa₃ is not an acidic amino acid.

According to the method of the invention, a polypeptide that has atleast one copy of a desired peptide is recombinantly produced. Theproduction may be of a soluble polypeptide or may be an inclusion bodypreparation containing at least a substantially insoluble mass ofpolypeptide. Next, the polypeptide is proteolytically cleaved usingclostripain to produce the desired peptide. The proteolytic reaction canbe performed on the solubilzed cellular contents in situations where thepolypeptide is soluble. Or, it may be performed on crude preparations ofinclusion bodies. In either situation, separation steps prior to orfollowing the enzymatic cleavage may be employed. Use of varyingconcentrations of urea in the medium containing the crude cellularcontents or inclusion bodies in optional combination with suchseparation steps may also be employed. A reaction vessel can also beused that permits continuous recovery and separation of the peptide awayfrom the uncleaved polypeptide and the clostripain. Use of such a methodproduces large amounts of pure peptide in essentially one step,eliminating numerous processing steps typically used in currentlyavailable procedures.

Clostripain can be used to cleave purified or impure preparations of thepolypeptide. The precursor polypeptide can be in solution or it can bean insoluble mass. For example, the precursor polypeptide can be in apreparation of inclusion bodies that becomes soluble in the reactionmixture. According to the invention, clostripain is active in highlevels of reagents that are commonly used to solubilize proteins. Forexample, clostripain is active in high levels of urea. Therefore,concentrations of urea ranging up to about 8M can readily be used in theclostripain cleavage reaction.

Little purification of the polypeptide is required when an inclusionbody preparation of the polypeptide is used as a substrate forclostripain cleavage. Essentially, host cells having a recombinantnucleic acid encoding the polypeptide are grown under conditions thatpermit expression of the polypeptide. Cells are grown to high celldensities, then collected, washed and broken open, for example, bysonication. Inclusion bodies are then collected, washed in water andemployed without further purification.

Up to about 8M urea can be used to solubilized insoluble precursorpolypeptides, for example, inclusion body preparations of precursorpolypeptides. The amount of urea employed can vary depending on theprecursor polypeptide. For example, about 0 M to about 8 M urea can beemployed in the clostripain reaction mixture to solubilize the precursorpolypeptide. Preferred concentrations of urea are about 4 M urea toabout 8 M urea.

Urea can also be used in the clostripain reaction. Concentrations of upto 8 M urea can be used in the clostripain cleavage. Preferredconcentrations of urea are about 0.0 to about 4 M urea. More preferredconcentrations of urea are about 0.0 to about 1.0 M urea. Even morepreferred concentrations of urea are about 0.0 to about 0.5 M urea.

In some cases, it may be preferable to remove the urea before cleavagewith clostripain. In such cases, urea may be removed by dialysis, gelfiltration, tangential flow filtration (TFF), numerous otherchromatographic methods, and the like.

Moreover, according to the invention, the cleavage reaction conditionscan be modified so that clostripain will have an even strongerpreference for cleavage at sites having formula I. Several factors canbe modified or implemented to obtain the desired product. Thus, byadjusting the pH and adding organic solvents, such as ethanol oracetonitrile, or by using a selected amount of enzyme relative toprecursor polypeptide and/or by using selected reaction times and/or bycontinuously removing the peptide as it is formed, cleavage at undesiredsites can be avoided.

Appropriate inorganic or organic buffers can be used to control the pHof the cleavage reaction. Such buffers include phosphate, Tris, glycine,HEPES and the like. The pH of the reaction can vary between pH 4 and pH12. However, a pH range between pH 6 and pH 10 is preferred. Foramidation, a pH range between 8.5 and 10.5 is preferred. While forhydrolysis, a pH range between 6 and 7 is preferred. When the cleavageis performed on precursor polypeptides in the absence or presence ofsignificant amounts of urea, pH values ranging from about 6.0 to about6.9 are preferred.

The activity of the clostripain enzyme has surprisingly been found to beinfluenced by the presence of organic solvents. For example, ethanol andacetonitrile may be used to increase the rate of substrate cleavage aswell as the overall yield of product formed from the cleavage of aprecursor polypeptide. Another surprising result is that organicsolvents influence the cleavage specificity of clostripain. Thus, thepresence of an organic solvent can dramatically influence thepreferential hydrolysis of one cleavage site in a precursor polypeptiderelative to another cleavage site within the same precursor polypeptide.This characteristic of clostripain can be exploited to design precursorpolypeptides that are rapidly and preferentially cleaved at specificsites within the precursor polypeptide.

The clostripain enzyme can be activated at similar pH ranges. A suitablebuffer substance, for example phosphate, Tris, HEPES, glycine and thelike, can be added to maintain the pH.

The concentration of the precursor polypeptide employed during thecleavage is, for example, between 0.01 mg/ml and 100 mg/ml, preferablybetween 0.1 mg/ml and 20 mg/ml. The ratio of polypeptide to clostripainis, in mg to units about 1:0.01 to about 1:1,000, preferably about 1:0.1to about 1:50. The temperature of the reaction can also be varied over awide range and may depend upon the selected reaction conditions. Such arange can be between 0° C. and +80° C. A preferred temperature range isgenerally between +5° C. and +60° C. Amidation is preferably conductedat a temperature between 5° C. and 60° C., and is more preferablyconducted at a temperature between 35° C. and 60° C., and is mostpreferably conducted at 45° C. Hydrolysis is preferably conducted at atemperature between 20° C. and 30° C., and more preferably is conductedat 25° C.

The time required for the conversion of the precursor polypeptides intothe peptides of interest can vary and one of skill in the art canreadily ascertain an appropriate reaction time. For example, thereaction time can vary between about 1 minute and 48 hours can beutilized. However, a reaction time of between 0.5 h and 6 h ispreferred. A reaction time of 0.5 h and 2 hours is more preferred. Insome embodiments, the reaction mixture is preferably placed in areaction vessel that permits continuous removal of the peptide product.For example, the reaction vessel can have a filter that permits thepeptide product of interest to pass through but that retains theprecursor polypeptide and the clostripain. An example of an appropriatefiltration system is tangential flow filtration (TFF). Reaction buffer,substrate and other components of the reaction mixture can be addedbatchwise or continuously as the peptide is removed and the reactionvolume is lost.

The enzyme can be activated before use in a suitable manner in thepresence of a mercaptan. Mercaptans suitable for activation arecompounds containing SH groups. Examples of such activating compoundsinclude DTT, DTE, mercaptoethanol, thioglycolic acid or cysteine.Cysteine is preferably used. The concentration of the mercaptan can alsovary. In general, concentrations between about 0.01 mM and 50 mM areuseful. Preferred mercaptan concentrations include concentrationsbetween about 0.05 mM and 5 mM. More preferred mercaptan concentrationsare between about 0.5 mM and 2 mM. The activation temperature can bebetween 0° C. and 80° C. Preferably the activation temperature can bebetween 0° C. and 40° C., more preferably the activation temperature isbetween 0° C. and 30° C. Most preferably the activation temperature isbetween 15° C. and 25° C.

Clostripain can be purchased from commercially available sources orprepared from microorganisms. Natural and recombinant clostripain isavailable. For example, natural clostripain can be prepared fromClostridia bacteria by cultivating the bacteria until clostripainaccumulates in the nutrient medium. Clostridia used for producingclostripain include, for example, Clostridium histolyticum, especiallyClostridium histolyticum DSM 627. Culturing is carried outanaerobically, singly or in mixed culture, for example, in non-agitatedculture in the absence of oxygen or in fermenters. Where appropriatenitrogen, inert gases or other gases apart from oxygen can be introducedinto the culture. The fermentation is carried out in a temperature rangefrom about 10° to 45° C., preferably about 25° to 40° C., especially 30°to 38° C. Fermentation takes place in a pH range between 5 and 8.5,preferably between 5.5 and 8. Under these conditions, the culture brothgenerally shows a detectable accumulation of the enzyme after 1 to 3days. The synthesis of clostripain starts in the late log phase andreaches its maximum in the stationary phase of growth. The production ofthe enzyme can be followed by means of activity assays (Mitchell, Meth.of Enzymol., 47:165 (1977)).

The nutrient solution used for producing clostripain can contain 0.2 to6%, preferably 0.5 to 3%, of organic nitrogen compounds, and inorganicsalts. Suitable organic nitrogen compounds are: amino acids, peptones,also meat extracts, milled seeds, for example of corn, wheat, beans,soybean or the cotton plant, distillation residues from alcoholproduction, meat meals or yeast extracts. Examples of inorganic saltsthat the nutrient solution can contain are chlorides, carbonates,sulfates or phosphates of the alkali metals or alkaline earth metals,iron, zinc and manganese, but also ammonium salts and nitrates.

Clostripain can be purified by classical processes, for example byammonium sulfate precipitation, ion exchange or gel permeationchromatography. Clostripain can also be produced recombinantly andthereafter purified according to standard methods.

Peptides of Interest Serving as Substrates According to the Invention

Almost any peptide can be formed by the methods of the invention.Peptides with an arginine at their C-terminus can readily be cleavedfrom a polypeptide containing end-to-end copies of the peptide. Peptideswith one or more internal arginine residues can also be made byemploying the teachings of the invention on which arginine-containingsites are favored for cleavage. Peptides having C-terminal amino acidsother than arginine can be produced by placing a clostripain cleavagesite within the polypeptide at the N-terminus of the peptide ofinterest. This latter technique produces the single copy desired peptideand employs a recombinantly expressed polypeptide having a discardablepeptide sequence at the N-terminal side of the desired peptide.

Clostripain is generally perceived to be an “arginine” or an“arginine/lysine” protease, meaning that clostripain cleavespolypeptides on the carboxyl side of arginine and/or lysine amino acidresidues. However, according to the invention, clostripain has evengreater specificity, particularly under the reaction conditions providesherein. Hence, peptides with internal lysine and arginine residues canbe made by the procedures of the invention.

Moreover, the construction of the polypeptide can be manipulated so thatthe peptide of interest is present at the C-terminus of the polypeptideand a clostripain cleavage site is at the N-terminus of the peptide ofinterest. Hence, when cleavage is performed on a polypeptide containingsuch a C-terminal peptide, the peptide is readily released. Using such aprecursor polypeptide, peptides with any C-terminal residue can beformed.

According to the invention, peptides having one or more internalarginine residues can still be selectively cleaved at their termini sothat a functional, full-length peptide can be recovered. This enhancedselectivity is achieved by recognition that clostripain preferentiallycleaves a polypeptide having a sequence as shown in Formula I, where thecleavage occurs at a peptide bond after amino acid Xaa₂:Xaa₁-Xaa₂-Xaa₃  (I)

-   -   wherein        -   Xaa₁ aspartic acid, glycine, proline or glutamic acid;        -   Xaa₂ is arginine; and        -   Xaa₃ is not an acidic amino acid.

Hence, a peptide of the Formula Xaa₃-Peptide₁-Xaa₁-Xaa₂, can readily beexcised from a polypeptide having end-to-end concatemers of the peptide,when Xaa₁, Xaa₂, and Xaa₃ are as described above. Peptide₁ refers to apeptidyl entity that is unique to the selected peptide of interest.Hence, Peptide₁ has any amino acid sequence that is selected by one ofskill in the art. An example of such a polypeptide with end-to-endconcatemers of the peptide of interest has Formula II:(Xaa₃-Peptide₁-Xaa₁-Xaa₂)_(n)-Xaa₃-Peptide₁-Xaa₁-Xaa₂  (II)

-   -   wherein        -   the peptide produced comprises Xaa₃-Peptide₁-Xaa₁-Xaa₂;        -   the desired GLP-2 peptides have the formula Xaa₃-Peptide₁;        -   n is an integer ranging from 0 to 50;        -   Xaa₁ is aspartic acid, glycine, proline or glutamic acid;        -   Xaa₂ is arginine; and        -   Xaa₃ is not an acidic amino acid.

However, the invention is not limited to cleavage of polypeptides havingend-to-end concatemers of a peptide of interest. The invention alsoprovides methods of making large amounts of a peptide that is present asa single copy within a polypeptide. This aspect of the invention enablesthe production of a single copy desired peptide having virtually anyamino acid sequence and one not having an arginine at the C-terminus,such as the desired GLP-2 peptides of the invention. That is, theinvention provides methods of making large amounts of peptides of theFormula, Xaa₃-Peptide₁, which do not have a C-terminal lysine orarginine. A cleavable peptide linker can be attached onto the peptide(e.g., Linker-Xaa₃-Peptide₁) to generate an N-terminal cleavage site forgenerating peptides of interest that have no C-terminal arginine orlysine. The Linker has a C-terminal Xaa₁-Xaa₂ sequence that directscleavage to the junction between the C-terminal Xaa₂ residue of theLinker and the Xaa₃ N-terminal residue of the peptide. Hence, peptidesof the Formula, Xaa₃-Peptide₁, that have C-terminal acidic, aliphatic oraromatic amino acids can readily be made by the present methods.

Cleavage of a peptide of the Formula, Xaa₃-Peptide₁, from a polypeptidehaving at least one copy of the peptide relies upon the presence of asite that has Formula I (Xaa₁-Xaa₂-Xaa₃) at the junction between thepeptide and the attached Linker or polypeptide. The Xaa₃ amino acidforms the N-terminal end of the peptide and is not an acidic amino acidsequence. Polypeptides of Formula III can readily be cleaved byclostripain:(Linker-Xaa₁-Xaa₂-Xaa₃-Peptide₁)_(n)-Linker-Xaa₁-Xaa₂-Xaa₃-Peptide₁  FormulaIII

-   -   wherein        -   the peptide comprises Xaa₃-Peptide₁        -   n is an integer ranging from 0 to 50;        -   Xaa₁ is aspartic acid, glycine, proline or glutamic acid;        -   Xaa₂ is arginine; and        -   Xaa₃ is not an acidic amino acid.

Cleavage of a polypeptide of Formula III yields one molar equivalent ofthe Xaa₃-Peptide₁ and n molar equivalents of a polypeptide of thefollowing structure: Xaa₃-Peptide₁-Linker-Xaa₁-Xaa₂. While thispolypeptide may not have a specific utility after cleavage, many“unused” parts of the linker or the polypeptide do have specificpurposes. For example, the Xaa₁-Xaa₂ amino acids in the polypeptide arerecognized by and direct clostripain to cleave the Xaa₂-Xaa₃ peptidebond with specificity. As described in the section entitled “Precursorpolypeptides,” other parts of the polypeptide or the linker havespecific functions relating to the recombinant expression, translation,sub-cellular localization, etc. of the polypeptide within the host cell.

Almost any peptide of interest to one of skill in the art can be made bythe methods of the invention. In particular, preferred peptides ofinterest (desired peptides) include, for example, a GLP-2 glucagon-likepeptide. Different kinds of GLP-2 peptides can be made by the methods ofthe invention include, for example,

-   GLP-2(1-33) (SEQ ID NO:11), GLP-2(1-33) amide (SEQ ID NO:12),    GLP-2(1-33, A2G) (SEQ ID NO:13), GLP-2(1-33,A2G) amide (SEQ ID    NO:14), GLP-2(1-34) (SEQ ID NO:9), GLP-2(1-34)NH₂ (SEQ ID NO:10),    GLP-2(1-34)A2G (SEQ ID NO:15), GLP-2 (1-34)A2G-NH₂ (SEQ ID NO:16),    and the like. The sequences of such GLPs are provided in Table 1    along with their names and SEQ ID NO: (“NO:”).

TABLE 1 SEQ ID Name Sequence NO: GLP-2(1-34) HADGSFSDEMNTILDNLAARD 9FINWLIQTKITDR GLP-2(1-34)NH₂ HADGSFSDEMNTILDNLAARD 10 FINWLIQTKITDR-NH₂GLP-2(1-33) HADGSFSDEMNTILDNLAARD 11 FINWLIQTKITD GLP-2(1-33)-NH₂HADGSFSDEMNTILDNLAARD 12 FINWLIQTKITD-NH₂ GLP-2(1-33, A2G)HGDGSFSDEMNTILDNLAARD 13 FINWLIQTKITD GLP-2(1-33, A2G)-NH₂HGDGSFSDEMNTILDNLAARD 14 FINWLIQTKITD-NH₂ GLP-2(1-34)A2GHGDGSFSDEMNTILDNLAARD 15 FINWLIQTKITDR GLP-2(1-34)A2G-NH₂HGDGSFSDEMNTILDNLAARD  16 FINWLIQTKITDR-NH₂

The invention also contemplates peptide variants, mutations, andderivatives of the GLP-2 peptides described herein. Derivatives,mutations and variant peptides of the invention are derived from thereference peptide by deletion, substitution or addition of one or moreamino acids to the N-terminal and/or C-terminal end; deletion,substitution or addition of one or more amino acids at one or more siteswithin the peptide; or substitution of one or more amino acids at one ormore sites of peptide. Thus, the GLP-2 peptides of the invention may bealtered in various ways including amino acid substitutions, deletions,truncations, and insertions. The invention also includes the GLP-2peptides, analogs, variants, modifications, additions, substitutions,deletions and the like disclosed in U.S. Pat. Nos. 5,990,077 and6,184,201

Such variant and derivative polypeptides may result, for example, fromhuman manipulation. Methods for such manipulations are generally knownin the art. For example, amino acid sequence variants of thepolypeptides can be prepared by mutations in the DNA. Methods formutagenesis and nucleotide sequence alterations are well-known in theart. See, for example, Kunkel, Proc. Natl. Acad. Sci. USA, 82:488(1985); Kunkel et al., Methods in Enzymol., 154:367 (1987); U.S. Pat.No. 4,873,192; Walker and Gaastra, eds., Techniques in MolecularBiology, MacMillan Publishing Company, New York (1983) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al., Atlas of ProteinSequence and Structure, Natl. Biomed. Res. Found., Washington, C.D.(1978), herein incorporated by reference.

Precursor Polypeptides

Any precursor polypeptide containing one or more copies of a peptide ofinterest (desired peptide) and a Formula I sequence at one or both endsof that peptide can be utilized as a substrate for the clostripaincleavage methods of the invention. One of skill in the art can readilydesign many such precursor polypeptides. While the peptide of interestmay form a substantial portion of the precursor polypeptide, thepolypeptide may also have additional peptide segments unrelated to thepeptide sequence of interest. Additional peptide segments can provideany function desired by one of skill in the art.

One example of an additional peptide segment that can be present in theprecursor polypeptide is a “Tag” that provides greater levels ofprecursor polypeptide production in cells. Numerous tag sequences areknown in the art. In the present invention, bacterial tag sequences arepreferred. Such tag sequences may be obtained from gene 10 bacteriophageT7, and the gene encoding ompT. In one embodiment, a T7 tag is used thathas the amino acid sequence ASMTGGQQMGR (SEQ ID NO:17). In anotherembodiment, a T7 tag is used that has the amino acid sequenceMASMTGGQQMGR (SEQ ID NO:18).

The precursor polypeptide can also encode an “inclusion body leaderpartner” that is operably linked to the peptide of interest. Such aninclusion body leader partner may be linked to the amino-terminus, thecarboxyl-terminus or both termini of a precursor polypeptide. In oneexample, the inclusion body leader partner has an amino acid sequencecorresponding to: GSGQGQAQYLSASCVVFTNYSGDTASQVD (SEQ ID NO:19). Inanother embodiment, the inclusion body leader partner is a part of theDrosophila vestigial polypeptide (“Vg”), having sequenceGSGQGQAQYLAASLVVF TNYSGDTASQ VDVNGPRAMVD (SEQ ID NO:20). In anotherembodiment, the inclusion body leader partner is a part of polyhedrinpolypeptide (“Ph”), having sequence GSAEEEEILLEVSLVFKVKEFAPDAPLFTGPAYVD(SEQ ID NO:21). Other inclusion body leader partners that can be usedinclude a part of the lactamase polypeptide, having sequenceSIQHFRVALIPFFAAFSLPVFA (SEQ ID NO:22). Upon expression of thepolypeptide, an attached inclusion body leader partner causes thepolypeptide to form inclusion bodies within the bacterial host cell.Other inclusion body leader partners can be identified, for example, bylinking a test inclusion body leader partner to a polypeptide construct.The resulting inclusion body leader partner-polypeptide construct thenwould be tested to determine whether it will form an inclusion bodywithin a cell.

The amino acid sequence of an inclusion body leader partner can bealtered to produce inclusion bodies that facilitate isolation ofinclusion bodies that are formed, thereby allowing an attachedpolypeptide to be purified more easily. For example, the inclusion bodyleader partner may be altered to produce inclusion bodies that are moreor less soluble under a certain set of conditions. Those of skill in theart realize that solubility is dependent on a number of variables thatinclude, but are not limited to, pH, temperature, salt concentration,protein concentration and the hydrophilicity or hydrophobicity of theamino acids in the protein. Thus, an inclusion body leader partner ofthe invention may be altered to produce an inclusion body having desiredsolubility under differing conditions.

An inclusion body leader partner may also be altered to produceinclusion bodies that contain polypeptide constructs having greater orlesser self-association. Self-association refers to the strength of theinteraction between two or more polypeptides that form an inclusionbody. Such self-association may be determined though use of a variety ofknown methods used to measure protein-protein interactions. Such methodsare known in the art and have been described. Freifelder, PhysicalBiochemistry: Applications to Biochemistry and Molecular Biology, W.H.Freeman and Co., 2nd edition, New York, N.Y. (1982).

Self-adhesion can be used to produce inclusion bodies that exhibitvarying stability to purification. For example, greater self-adhesionmay be desirable to stabilize inclusion bodies against dissociation ininstances where harsh conditions are used to isolate the inclusionbodies from a cell. Such conditions may be encountered if inclusionbodies are being isolated from cells having thick cell walls. However,where mild conditions are used to isolate the inclusion bodies, lessself-adhesion may be desirable as it may allow the polypeptideconstructs composing the inclusion body to be more readily solubilizedor processed. Accordingly, an inclusion body leader partner of theinvention may be altered to provide a desired level of self-adhesion fora given set of conditions.

The precursor polypeptide can also encode one or more “cleavable peptidelinkers” that can flank one or more copies of the peptide of interest.Such a cleavable peptide linker provides a convenient clostripaincleavage site adjacent to a peptide of interest, and allows a peptidethat does not naturally begin or end with an arginine or lysine to beexcised with clostripain. Convenient cleavable peptide linkers includeshort peptidyl sequences having a C-terminal Xaa₁-Xaa₂ sequence, forexample, a Linker-Xaa₁-Xaa₂ sequence, wherein Xaa₁ is aspartic acid,glycine, proline or glutamic acid, and Xaa₂ is arginine. The Xaa₁-Xaa₂sequence directs cleavage to the junction between the C-terminal Xaa₂residue of the linker and a Xaa₃ residue on the N-terminus of thepeptide.

A cleavable peptide linker can have the following Formula IV:(Peptide₅)_(m)-Xaa₁-Xaa₂  IVwherein:

n and m are separately an integer ranging from 0 to 50;

Xaa₁ is aspartic acid, glycine, proline or glutamic acid; and

Xaa₂ is arginine; and

Peptide₅ is any single or multiple amino acid residue.

In some embodiments, use of Peptide₅ as proline is preferred.

Many cleavable peptide linker sequences can readily be developed andused by one of skill in the art. A few examples of convenient cleavablepeptide linker sequences are provided below.

Ala-Phe-Leu-Gly-Pro-Gly-Asp-Arg (SEQ ID NO: 23) Val-Asp-Asp-Arg(SEQ ID NO: 24) Gly-Ser-Asp-Arg (SEQ ID NO: 25) Ile-Thr-Asp-Arg(SEQ ID NO: 26) Pro-Gly-Asp-Arg. (SEQ ID NO: 27)

Other amino acids, peptides, or polypeptides selected by one of skill inthe art can also be included in the precursor polypeptide.

GLP-2 Polypeptides

In one embodiment of the invention, the polypeptide can encode one ormore copies of GLP-2.

Examples of multi-copy GLP-2 polypeptides include polypeptides havingthe following generalized structures:Tag-Linker-[GLP-2(1-34)]_(q)  VIWhere GLP-2 (1-34) has SEQ ID NO:9 and q is an integer of about 2 toabout 20. A preferred value for q is about 6. The Linker is preferablyPeptide₅-Asp-Arg or Xaa₄-Xaa₅-Asp-Arg-Arg. Tag is a translationinitiation sequence, for example, SEQ ID NO: 18. A multi-copy GLP-2polypeptide of this generalized structure with q equal to 6 and withLinker as Peptide₅-Asp-Arg (GSDR) has the following sequence:

(SEQ ID NO: 29) MASMTGGQQMGR-GSDR- HADGSFSDEMNTILDNLAARDFINWLIQTKITDR-HADGSFSDEMNTILDNLAARDFINWLIQTKITDR- HADGSFSDEMNTILDNLAARDFINWLIQTKITDR-HADGSFSDEMNTILDNLAARDFINWLIQTKITDR- HADGSFSDEMNTILDNLAARDFINWLIQTKITDR-HADGSFSDEMNTILDNLAARDFINWLIQTKITDR. No cleavable peptide linkers are needed between the GLP-2 six peptidespresent within this precursor polypeptide because GLP-2(1-34) has anAsp-Arg sequence at its C-terminus.

In another embodiment of the invention, the polypeptide can encode acopy of GLP-2.

Examples of such a GLP-2 polypeptide include polypeptides having thefollowing generalized structures:Tag-Linker-[GLP-2(1-33)]  VIWhere GLP-2(1-33) has SEQ ID NO: 11. The Linker is preferablyPeptide₅-Asp-Arg or Peptide_(s)-Asp-Arg-Arg. Tag is a translationinitiation sequence, for example, SEQ ID NO: 17 or 18. A multi-copyGLP-2 polypeptide of this generalized structure with Linker asPeptide₅-Asp-Arg (GSDR) has the following sequence:

(SEQ ID NO:28) MASMTGGQQMGR-GSDR- HADGSFSDEMNTILDNLAARDFINWLIQTKITD.

One of skill in the art can modify or mutate these GLP-2 polypeptidesequences as desired so long as the aspartic acid at position 21 ofGLP-2 (HADGSFSDEMNTILDN LAARDFINWLIQTKITDR, SEQ ID NO:9) or GLP-2(HADGSFSDEMNTILDN LAARDFINWLIQTKITD, SEQ ID NO:11) is not changed. Thisaspartic acid is on the C-terminal side of an arginine and is thereforeat position Xaa₃ in the clostripain cleavage site. As described, Xaa₃should not be an acidic amino acid when clostripain cleavage is desired.However, in the GLP-2 polypeptides described above, an acidic amino acidat position 21 (Asp-21) protects against cleavage at the internalarginine. Recognition that Asp-21 protects against cleavage allows afull-length GLP-2 peptide to be produced in far larger amounts than aGLP-2 fragment containing only amino acids 1-19.

One mutation that can be made is a substitution of glycine for alanineat position 2 of the GLP-2 peptide, to produce GLP-2(1-34)A2G having SEQID NO:15, or GLP-2(1-33, A2G) having SEQ ID NO:13. This amino acidsubstitution of glycine for alanine produces a GLP-2 peptide that lacksa recognition site for a eukaryotic endopeptidase that might degrade thepeptide upon administration to a mammal. Hence, a GLP-2(1-33,A2G) orGLP-2(1-34,A2G) peptide can have a longer half-life in vivo than theGLP-2(1-33) or GLP-2(1-34) peptide.

Amidation Conditions

When clipped from a multicopy polypeptide under normal hydrolysisconditions, recombinant GLP-2 has a C-terminal carboxyl group. However,an amidated C-terminus is preferred for use in mammals. Clostripain canbe used to amidate the C-terminal residue to make an amidatedrecombinant GLP-2 by adjusting the conditions to increase the amount ofamide formation. However, the recombinant GLP-2 amide itself becomes asubstrate for hydrolysis as it is formed. To solve this problem, atangential flow filtration in combination with the enzyme reaction isused. Clostripain simultaneously cleaves multicopy peptide constructsand amidates the C-terminal residue of the single copy cleaved peptide.Use of tangential flow filtration during the enzymatic reaction toremove the amidated peptide produces that peptide in high yield.

For example, use of a 10K diafiltration/tangential flow filtrationmembrane will enhance the reaction yield. Undigested peptide constructand clostripain are retained on the retentate side of the membrane. Thesingle copy cleaved GLP-2 passes through the membrane. Continuedexposure of GLP-2 amide to clostripain will result in loss of the amideto OH. Continual removal of amide through the membrane will reduce thisunwanted side reaction. Smaller pore sized membranes were not asefficient at removing the newly formed GLP-2 amide during the reactiontime course.

Clostripain, like other proteases, will perform transpeptidationreactions in the presence of a nucleophile other than water. Ammonia orother amines can be used as the nucleophile. A polypeptide that hadthree copies of the GLP-2 peptide was used as a substrate. Thepolypeptide had a leader sequence as well.

Reaction conditions will enhance the transpeptidation reaction relativeto hydrolysis for this particular polypeptide construct. Urea in theclostripain reaction maintains peptide solubility and minimizes membranefouling. The clostripain digestion/amidation reaction will toleratehigher urea concentrations. The amount of clostripain can be varied toshorten or lengthen the overall reaction time. Fresh buffer can be addedto maintain constant volume or after volume reduction. However, caremust be maintained to ensure the minimum volume of liquid remains inplace to prevent foaming.

Production of Precursor Polypeptides

A) DNA Constructs and Expression Cassettes

Precursor polypeptides are produced in any convenient manner, forexample, by using a recombinant nucleic acid that encodes the desiredprecursor polypeptide. Nucleic acids encoding the precursor polypeptidesof the invention can be inserted into convenient vectors fortransformation of an appropriate host cell. Those of skill in the artcan readily obtain and clone nucleic acids encoding a selected precursorpolypeptide into a variety of commercially available plasmids. Oneexample of a useful plasmid vector is the pET series of plasmids(Stratagene, La Jolla, Calif.). After insertion of the selected nucleicacid into an appropriate vector, the vector can be introduced into ahost cell, preferably a bacterial host cell.

Nucleic acid constructs and expression cassettes can be created throughuse of recombinant methods that are available in the art. (Sambrook andRussell, Molecular Cloning: A Laboratory Manual, 3rd edition (Jan. 15,2001) Cold Spring Harbor Laboratory Press, ISBN: 0879695765; Ausubel etal., Current Protocols in Molecular Biology, Green Publishing Associatesand Wiley Interscience, NY (1989)). Generally, recombinant methodsinvolve preparation of a desired DNA fragment and ligation of that DNAfragment into a preselected position in another DNA vector, such as aplasmid.

In a typical example, a desired DNA fragment is first obtained bysynthesizing and/or digesting a DNA that contains the desired DNAfragment with one or more restriction enzymes that cut on both sides ofthe desired DNA fragment. The restriction enzymes may leave a “blunt”end or a “sticky” end. A “blunt” end means that the end of a DNAfragment does not contain a region of single-stranded DNA. A DNAfragment having a “sticky” end means that the end of the DNA fragmenthas a region of single-stranded DNA. The sticky end may have a 5′ or a3′ overhang. Numerous restriction enzymes are commercially available andconditions for their use are also well-known. (USB, Cleveland, Ohio; NewEngland Biolabs, Beverly, Mass.).

The digested DNA fragments may be extracted according to known methods,such as phenol/chloroform extraction, to produce DNA fragments free fromrestriction enzymes. The restriction enzymes may also be inactivatedwith heat or other suitable means. Alternatively, a desired DNA fragmentmay be isolated away from additional nucleic acid sequences andrestriction enzymes through use of electrophoresis, such as agarose gelor polyacrylamide gel electrophoresis. Generally, agarose gelelectrophoresis is used to isolate large nucleic acid fragments whilepolyacrylamide gel electrophoresis is used to isolate small nucleic acidfragments. Such methods are used routinely to isolate DNA fragments. Theelectrophoresed DNA fragment can then be extracted from the gelfollowing electrophoresis through use of many known methods, such aselectoelution, column chromatography, or binding of glass beads. Manykits containing materials and methods for extraction and isolation ofDNA fragments are commercially available. (Qiagen, Venlo, Netherlands;Qbiogene, Carlsbad, Calif.).

The DNA segment into which the fragment is going to be inserted is thendigested with one or more restriction enzymes. Preferably, the DNAsegment is digested with the same restriction enzymes used to producethe desired DNA fragment. This will allow for directional insertion ofthe DNA fragment into the DNA segment based on the orientation of thecomplimentary ends. For example, if a DNA fragment is produced that hasan EcoRI site on its 5′ end and a BamHI site at the 3′ end, it may bedirectionally inserted into a DNA segment that has been digested withEcoRI and BamHI based on the complementarity of the ends of therespective DNAs. Alternatively, blunt ended cloning may be used if noconvenient restriction sites exist that allow for directional cloning.For example, the restriction enzyme BsaAI leaves DNA ends that do nothave a 5′ or 3′ overhang. Blunt ended cloning may be used to insert aDNA fragment into a DNA segment that was also digested with an enzymethat produces a blunt end. Additionally, DNA fragments and segments maybe digested with a restriction enzyme that produces an overhang and thentreated with an appropriate enzyme to produce a blunt end. Such enzymesinclude polymerases and exonucleases. Those of skill in the art know howto use such methods alone or in combination to selectively produce DNAfragments and segments that may be selectively combined.

A DNA fragment and a DNA segment can be combined though conducting aligation reaction. Ligation links two pieces of DNA through formation ofa phosphodiester bond between the two pieces of DNA. Generally, ligationof two or more pieces of DNA occurs through the action of the enzymeligase when the pieces of DNA are incubated with ligase underappropriate conditions. Ligase and methods and conditions for its useare well-known in the art and are commercially available.

The ligation reaction or a portion thereof is then used to transformcells to amplify the recombinant DNA formed, such as a plasmid having aninsert. Methods for introducing DNA into cells are well-known and aredisclosed herein.

Those of skill in the art recognize that many techniques for producingrecombinant nucleic acids can be used to produce an expression cassetteor nucleic acid construct of the invention.

B) Promoters

The expression cassette of the invention includes a promoter. Anypromoter able to direct transcription of the expression cassette may beused. Accordingly, many promoters may be included within the expressioncassette of the invention. Some useful promoters include, constitutivepromoters, inducible promoters, regulated promoters, cell specificpromoters, viral promoters, and synthetic promoters. A promoter is anucleotide sequence which controls expression of an operably linkednucleic acid sequence by providing a recognition site for RNApolymerase, and possibly other factors, required for propertranscription. A promoter includes a minimal promoter, consisting onlyof all basal elements needed for transcription initiation, such as aTATA-box and/or other sequences that serve to specify the site oftranscription initiation. A promoter may be obtained from a variety ofdifferent sources. For example, a promoter may be derived entirely froma native gene, be composed of different elements derived from differentpromoters found in nature, or be composed of nucleic acid sequences thatare entirely synthetic. A promoter may be derived from many differenttypes of organisms and tailored for use within a given cell.

Examples of Promoters Useful in Bacteria

For expression of a precursor polypeptide in a bacterium, an expressioncassette having a bacterial promoter will be used. A bacterial promoteris any DNA sequence capable of binding bacterial RNA polymerase andinitiating the downstream (3″) transcription of a coding sequence intomRNA. A promoter will have a transcription initiation region which isusually placed proximal to the 5′ end of the coding sequence. Thistranscription initiation region usually includes an RNA polymerasebinding site and a transcription initiation site. A second domain calledan operator may be present and overlap an adjacent RNA polymerasebinding site at which RNA synthesis begins. The operator permitsnegatively regulated (inducible) transcription, as a gene repressorprotein may bind the operator and thereby inhibit transcription of aspecific gene. Constitutive expression may occur in the absence ofnegative regulatory elements, such as the operator. In addition,positive regulation may be achieved by a gene activator protein bindingsequence, which, if present is usually proximal (5′) to the RNApolymerase binding sequence. An example of a gene activator protein isthe catabolite activator protein (CAP), which helps initiatetranscription of the lac operon in E. coli (Raibaud et al., Ann. Rev.Genet., 18:173 (1984)). Regulated expression may therefore be positiveor negative, thereby either enhancing or reducing transcription. Apreferred promoter is the YX chlorella virus promoter. (U.S. Pat. No.6,316,224).

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) (Chang etal., Nature, 198:1056 (1977), and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(tip) (Goeddel et al., Nuc. Acids Res., 8:4057 (1980); Yelverton et al.,Nuc. Acids Res., 9:731 (1981); U.S. Pat. No. 4,738,921; and EPO Publ.Nos. 036 776 and 121 775). The β-lactamase (bla) promoter system(Weissmann, “The cloning of interferon and other mistakes”, in:Interferon 3 (ed. I. Gresser), 1981), and bacteriophage lambda P_(L),(Shimatake et al., Nature, 292:128 (1981)) and T5 (U.S. Pat. No.4,689,406) promoter systems also provide useful promoter sequences.

Synthetic promoters which do not occur in nature also function asbacterial promoters. For example, transcription activation sequences ofone bacterial or bacteriophage promoter may be joined with the operonsequences of another bacterial or bacteriophage promoter, creating asynthetic hybrid promoter (U.S. Pat. No. 4,551,433). For example, thetac promoter is a hybrid trp-lac promoter comprised of both trp promoterand lac operon sequences that is regulated by the lac repressor (Amannet al., Gene, 25:167 (1983); de Boer et al., Proc. Natl. Acad. Sci. USA,80:21 (1983)). Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system (Studier et al., J.Mol. Biol., 189:113 (1986); Tabor et al., Proc. Natl. Acad. Sci. USA,82:1074 (1985)). In addition, a hybrid promoter can also be comprised ofa bacteriophage promoter and an E. coli operator region (EPO Publ. No.267 851).

Examples of Promoters Useful in Insect Cells

An expression cassette having a baculovirus promoter can be used forexpression of a precursor polypeptide in an insect cell. A baculoviruspromoter is any DNA sequence capable of binding a baculovirus RNApolymerase and initiating transcription of a coding sequence into mRNA.A promoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region usually includes an RNA polymerase binding site and atranscription initiation site. A second domain called an enhancer may bepresent and is usually distal to the structural gene. A baculoviruspromoter may be a regulated promoter or a constitutive promoter. Usefulpromoter sequences may be obtained from structural genes that aretranscribed at times late in a viral infection cycle. Examples includesequences derived from the gene encoding the baculoviral polyhedronprotein (Friesen et al., “The Regulation of Baculovirus GeneExpression”, in: The Molecular Biology of Baculoviruses (ed. WalterDoerfler), 1986; and EPO Publ. Nos. 127 839 and 155 476) and the geneencoding the baculoviral p10 protein (Vlak et al., J. Gen. Virol.,69:765 (1988)).

Examples of Promoters Useful in Yeast

Promoters that are functional in yeast are known to those of ordinaryskill in the art. In addition to an RNA polymerase binding site and atranscription initiation site, a yeast promoter may also have a secondregion called an upstream activator sequence. The upstream activatorsequence permits regulated expression that may be induced. Constitutiveexpression occurs in the absence of an upstream activator sequence.Regulated expression may be either positive or negative, thereby eitherenhancing or reducing transcription.

Promoters for use in yeast may be obtained from yeast genes that encodeenzymes active in metabolic pathways. Examples of such genes includealcohol dehydrogenase (ADH) (EPO Publ. No. 284 044), enolase,glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphatedehydrogenase (GAP or GAPDH), hexokinase,phosphofructokinase, 3-phosphoglyceratemutase, and pyruvate kinase(PyK). (EPO Publ. No. 329 203). The yeast PHOS gene, encoding acidphosphatase, also provides useful promoter sequences. (Myanohara et al.,Proc. Natl. Acad. Sci. USA, 80:1 (1983).

Synthetic promoters which do not occur in nature may also be used forexpression in yeast. For example, upstream activator sequences from oneyeast promoter may be joined with the transcription activation region ofanother yeast promoter, creating a synthetic hybrid promoter. Examplesof such hybrid promoters include the ADH regulatory sequence linked tothe GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and4,880,734). Other examples of hybrid promoters include promoters whichconsist of the regulatory sequences of either the ADH2, GAL4, GAL10, orPHOS genes, combined with the transcriptional activation region of aglycolytic enzyme gene such as GAP or PyK (EPO Publ. No. 164 556).Furthermore, a yeast promoter can include naturally occurring promotersof non-yeast origin that have the ability to bind yeast RNA polymeraseand initiate transcription. Examples of such promoters are known in theart. (Cohen et al., Proc. Natl. Acad. Sci. USA, 77:1078 (1980); Henikoffet al., Nature, 283:835 (1981); Hollenberg et al., Curr. TopicsMicrobiol. Immunol., 96:119 (1981); Hollenberg et al., “The Expressionof Bacterial Antibiotic Resistance Genes in the Yeast Saccharomycescerevisiae”, in: Plasmids of Medical, Environmental and CommercialImportance (eds. K. N. Timmis and A. Puhler), 1979; Mercerau-Puigalon etal., Gene, 11:163 (1980); Panthier et al., Curr. Genet., 2:109 (1980)).

Examples of Promoters Useful in Mammalian Cells

Many mammalian promoters are known in the art that may be used inconjunction with the expression cassette of the invention. Mammalianpromoters often have a transcription initiating region, which is usuallyplaced proximal to the 5′ end of the coding sequence, and a TATA-box,usually located 25-30 base pairs (bp) upstream of the transcriptioninitiation site. The TATA-box is thought to direct RNA polymerase II tobegin RNA synthesis at the correct site. A mammalian promoter may alsocontain an upstream promoter element, usually located within 100 to 200bp upstream of the TATA-box. An upstream promoter element determines therate at which transcription is initiated and can act in eitherorientation (Sambrook et al., “Expression of Cloned Genes in MammalianCells”, in: Molecular Cloning: A Laboratory Manual, 2nd ed., 1989).

Mammalian viral genes are often highly expressed and have a broad hostrange; therefore sequences encoding mammalian viral genes often provideuseful promoter sequences. Examples include the SV40 early promoter,mouse mammary tumour virus LTR promoter, adenovirus major late promoter(Ad MLP), and herpes simplex virus promoter. In addition, sequencesderived from non-viral genes, such as the murine metallothioneih gene,also provide useful promoter sequences. Expression may be eitherconstitutive or regulated.

A mammalian promoter may also be associated with an enhancer. Thepresence of an enhancer will usually increase transcription from anassociated promoter. An enhancer is a regulatory DNA sequence that canstimulate transcription up to 1000-fold when linked to homologous orheterologous promoters, with synthesis beginning at the normal RNA startsite. Enhancers are active when they are placed upstream or downstreamfrom the transcription initiation site, in either normal or flippedorientation, or at a distance of more than 1000 nucleotides from thepromoter. (Maniatis et al., Science, 236:1237 (1987)); Alberts et al.,Molecular Biology of the Cell, 2nd ed., 1989). Enhancer elements derivedfrom viruses are often times useful, because they usually have a broadhost range. Examples include the SV40 early gene enhancer (Dijkema etal., EMBO J., 4:761 (1985)) and the enhancer/promoters derived from thelong terminal repeat (LTR) of the Rous Sarcoma Virus (Gorman et al.,Proc. Natl. Acad. Sci. USA, 79:6777 (1982b)) and from humancytomegalovirus (Boshart et al., Cell, 41:521 (1985)). Additionally,some enhancers are regulatable and become active only in the presence ofan inducer, such as a hormone or metal ion (Sassone-Corsi and Borelli,Trends Genet., 2:215 (1986); Maniatis et al., Science, 236:1237 (1987)).

It is understood that many promoters and associated regulatory elementsmay be used within the expression cassette of the invention totranscribe an encoded leader protein. The promoters described above areprovided merely as examples and are not to be considered as a completelist of promoters that are included within the scope of the invention.

C) Translation Initiation Sequence

The expression cassette of the invention may contain a nucleic acidsequence for increasing the translation efficiency of an mRNA encoding aprecursor polypeptide of the invention. Such increased translationserves to increase production of the leader protein. The presence of anefficient ribosome binding site is useful for gene expression inprokaryotes. In bacterial mRNA a conserved stretch of six nucleotides,the Shine-Dalgarno sequence, is usually found upstream of the initiatingAUG codon. (Shine et al., Nature, 254:34 (1975)). This sequence isthought to promote ribosome binding to the mRNA by base pairing betweenthe ribosome binding site and the 3′ end of Escherichia coli 16S rRNA.(Steitz et al., “Genetic signals and nucleotide sequences in messengerRNA”, in: Biological Regulation and Development: Gene Expression (ed. R.F. Goldberger), 1979)). Such a ribosome binding site, or operablederivatives thereof, are included within the expression cassette of theinvention.

A translation initiation sequence can be derived from any expressedEscherichia coli gene and can be used within an expression cassette ofthe invention. Preferably the gene is a highly expressed gene. Atranslation initiation sequence can be obtained via standard recombinantmethods, synthetic techniques, purification techniques, or combinationsthereof, which are all well-known. (Ausubel et al., Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,NY. (1989); Beaucage and Caruthers, Tetra. Letts., 22:1859 (1981);VanDevanter et al., Nucleic Acids Res., 12:6159 (1984). Alternatively,translational start sequences can be obtained from numerous commercialvendors. (Operon Technologies; Life Technologies Inc, Gaithersburg,Md.). In a preferred embodiment, the T7 leader sequence is used. The T7leader sequence is derived from the highly expressed 17 Gene 10 cistron.Other examples of translation initiation sequences include, but are notlimited to, the maltose-binding protein (Mal E gene) start sequence(Guan et al., Gene, 67:21 (1997)) present in the pMalc2 expressionvector (New England Biolabs, Beverly, Mass.) and the translationinitiation sequence for the following genes: thioredoxin gene (Novagen,Madison, Wis.), Glutathione-S-transferase gene (Pharmacia, Piscataway,N.J.), β-galactosidase gene, chloramphenicol acetyltransferase gene andE. coli Trp E gene (Ausubel et al., 1989, Current Protocols in MolecularBiology, Chapter 16, Green Publishing Associates and Wiley Interscience,NY).

Eucaryotic mRNA does not contain a Shine-Dalgarno sequence. Instead, theselection of the translational start codon is usually determined by itsproximity to the cap at the 5′ end of an mRNA. The nucleotidesimmediately surrounding the start codon in eucaryotic mRNA influence theefficiency of translation. Accordingly, one skilled in the art candetermine what nucleic acid sequences will increase translation of aprecursor polypeptide encoded by the expression cassette of theinvention. Such nucleic acid sequences are within the scope of theinvention.

D) Vectors

Vectors that may be used include, but are not limited to, those able tobe replicated in prokaryotes and eukaryotes. Vectors include, forexample, plasmids, phagemids, bacteriophages, viruses, cosmids, andF-factors. The invention includes any vector into which the expressioncassette of the invention may be inserted and replicated in vitro or invivo. Specific vectors may be used for specific cells types.Additionally, shuttle vectors may be used for cloning and replication inmore than one cell type. Such shuttle vectors are known in the art. Thenucleic acid constructs may be carried extrachromosomally within a hostcell or may be integrated into a host cell chromosome. Numerous examplesof vectors are known in the art and are commercially available.(Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rdedition (Jan. 15, 2001) Cold Spring Harbor Laboratory Press, ISBN:0879695765; New England Biolabs, Beverly, Mass.; Stratagene, La Jolla,Calif.; Promega, Madison, Wis.; ATCC, Rockville, Md.; CLONTECH, PaloAlto, Calif.; Invitrogen, Carlsbad, Calif.; Origene, Rockville, Md.;Sigma, St. Louis, Mo.; Pharmacia, Peapack, N.J.; USB, Cleveland, Ohio).These vectors also provide many promoters and other regulatory elementsthat those of skill in the art may include within the nucleic acidconstructs of the invention through use of known recombinant techniques.

Examples of Vectors Useful in Bacteria

A nucleic acid construct for use in a prokaryote host, such as bacteria,will preferably include a replication system allowing it to bemaintained in the host for expression or for cloning and amplification.In addition, a nucleic acid construct may be present in the cell ineither high or low copy number. Generally, about 5 to about 200, andusually about 10 to about 150 copies of a high copy number nucleic acidconstruct will be present within a host cell. A host containing a highcopy number plasmid will preferably contain at least about 10, and morepreferably at least about 20 plasmids. Generally, about 1 to 10, andusually about 1 to 4 copies of a low copy number nucleic acid constructwill be present in a host cell. The copy number of a nucleic acidconstruct may be controlled by selection of different origins ofreplication according to methods known in the art. Sambrook and Russell,Molecular Cloning: A Laboratory Manual, 3rd edition (Jan. 15, 2001) ColdSpring Harbor Laboratory Press, ISBN: 0879695765.

A nucleic acid construct containing an expression cassette can beintegrated into the genome of a bacterial host cell through use of anintegrating vector. Integrating vectors usually contain at least onesequence that is homologous to the bacterial chromosome that allows thevector to integrate. Integrations are thought to result fromrecombinations between homologous DNA in the vector and the bacterialchromosome. For example, integrating vectors constructed with DNA fromvarious Bacillus strains integrate into the Bacillus chromosome (EPOPubl. No. 127 328). Integrating vectors may also contain bacteriophageor transposon sequences.

Extrachromosomal and integrating nucleic acid constructs may containselectable markers to allow for the selection of bacterial strains thathave been transformed. Selectable markers can be expressed in thebacterial host and may include genes that render bacteria resistant todrugs such as ampicillin, chloramphenicol, erythromycin, kanamycin(neomycin), and tetracycline (Davies et al., Ann. Rev. Microbiol.,32:469, 1978). Selectable markers may also include biosynthetic genes,such as those in the histidine, tryptophan, and leucine biosyntheticpathways.

Numerous vectors, either extra-chromosomal or integrating vectors, havebeen developed for transformation into many bacteria. For example,vectors have been developed for the following bacteria: B. subtilis(Palva et al., Proc. Natl. Acad. Sci. USA, 79:5582, 1982; EPO Publ. Nos.036 259 and 063 953; PCT Publ. No. WO 84/04541), E. coli (Shimatake etal., Nature, 292:128, 1981; Amann et al., Gene, 40:183, 1985; Studier etal., J. Mol. Biol., 189:113, 1986; EPO Publ. Nos. 036 776, 136 829 and136 907), Streptococcus cremoris (Powell et al., Appl. Environ.Microbiol., 54:655, 1988); Streptococcus lividans (Powell et al., Appl.Environ. Microbiol., 54:655, 1988), and Streptomyces lividans (U.S. Pat.No. 4,745,056). Numerous vectors are also commercially available (NewEngland Biolabs, Beverly, Mass.; Stratagene, La Jolla, Calif.).

Examples of Vectors Useful in Yeast

Many vectors may be used to construct a nucleic acid construct thatcontains an expression cassette of the invention and that provides forthe expression of a precursor polypeptide in yeast. Such vectorsinclude, but are not limited to, plasmids and yeast artificialchromosomes. Preferably the vector has two replication systems, thusallowing it to be maintained, for example, in yeast for expression andin a prokaryotic host for cloning and amplification. Examples of suchyeast-bacteria shuttle vectors include YEp24 (Botstein, et al., Gene,8:17 (1979)), pCl/1 (Brake et al., Proc. Natl. Acad. Sci. USA, 81:4642(1984)), and YRp17 (Stinchcomb et al., J. Mol. Biol., 158:157 (1982)). Avector may be maintained within a host cell in either high or low copynumber. For example, a high copy number plasmid will generally have acopy number ranging from about 5 to about 200, and usually about 10 toabout 150. A host containing a high copy number plasmid will preferablyhave at least about 10, and more preferably at least about 20. Either ahigh or low copy number vector may be selected, depending upon theeffect of the vector and the precursor polypeptide on the host. (Brakeet al., Proc. Natl. Acad. Sci. USA, 81:4642 (1984)).

A nucleic acid construct may also be integrated into the yeast genomewith an integrating vector. Integrating vectors usually contain at leastone sequence homologous to a yeast chromosome that allows the vector tointegrate, and preferably contain two homologous sequences flanking anexpression cassette of the invention. Integrations appear to result fromrecombinations between homologous DNA in the vector and the yeastchromosome. (Orr-Weaver et al., Methods in Enzymol., 101:228 (1983)). Anintegrating vector may be directed to a specific locus in yeast byselecting the appropriate homologous sequence for inclusion in thevector. One or more nucleic acid constructs may integrate, which mayaffect the level of recombinant protein produced. (Rine et al., Proc.Natl. Acad. Sci. USA, 80:6750 (1983)). The chromosomal sequencesincluded in the vector can occur either as a single segment in thevector, which results in the integration of the entire vector, or twosegments homologous to adjacent segments in the chromosome and flankingan expression cassette included in the vector, which can result in thestable integration of only the expression cassette.

Extrachromosomal and integrating nucleic acid constructs may containselectable markers that allow for selection of yeast strains that havebeen transformed. Selectable markers may include, but are not limitedto, biosynthetic genes that can be expressed in the yeast host, such asADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, whichconfer resistance in yeast cells to tunicamycin and G418, respectively.In addition, a selectable marker may also provide yeast with the abilityto grow in the presence of toxic compounds, such as metal. For example,the presence of CUP1 allows yeast to grow in the presence of copperions. (Butt et al., Microbiol. Rev., 51:351 (1987)).

Many vectors have been developed for transformation into many yeasts.For example, vectors have been developed for the following yeasts:Candida albicans (Kurtz et al., Mol. Cell. Biol., 6:142 (1986)), Candidamaltose (Kunze et al., J. Basic Microbiol., 25:141 (1985)), Hansenulapolymorpha (Gleeson et al., J. Gen. Microbiol., 132:3459 (1986);Roggenkamp et al., Mol. Gen. Genet., 202:302 (1986), Kluyveromycesfragilis (Das et at., J. Bacteriol., 158: 1165 (1984)), Kluyveromyceslactis (De Louvencourt et al., J. Bacteriol., 154:737 (1983); van denBerg et al., Bio/Technology, 8:135 (1990)), Pichia guillerimondii (Kunzeet al., J. Basic Microbiol., 25:141 (1985)), Pichia pastoris (Cregg etal., Mol. Cell. Biol., 5:3376, 1985; U.S. Pat. Nos. 4,837,148 and4,929,555), Saccharomyces cerevisiae (Hinnen et al., Proc. Natl. Acad.Sci. USA, 75:1929 (1978); Ito et al., J. Bacteriol., 153:163 (1983)),Schizosaccharomyces pombe (Beach and Nurse, Nature 300:706 (1981)), andYarrowia lipolytica (Davidow et al., Curr. Genet., 10:39 (1985);Gaillardin et al., Curr. Genet., 10:49 (1985)).

Examples of Vectors Useful in Insect Cells

Baculovirus vectors have been developed for infection into severalinsect cells and may be used to produce nucleic acid constructs thatcontain an expression cassette of the invention. For example,recombinant baculoviruses have been developed for Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni (PCT Pub. No. WO 89/046699; Carbonell etal., J. Virol., 56:153 (1985); Wright, Nature, 321: 718 (1986); Smith etal., Mol. Cell. Biol., 3: 2156 (1983); and see generally, Fraser et al.,In Vitro Cell. Dev. Biol., 25:225 (1989)). Such a baculovirus vector maybe used to introduce an expression cassette into an insect and providefor the expression of a precursor polypeptide within the insect cell.

Methods to form a nucleic acid construct having an expression cassetteof the invention inserted into a baculovirus vector are well-known inthe art. Briefly, an expression cassette of the invention is insertedinto a transfer vector, usually a bacterial plasmid which contains afragment of the baculovirus genome, through use of common recombinantmethods. The plasmid may also contain a polyhedrin polyadenylationsignal (Miller et al., Ann. Rev. Microbiol., 42:177 (1988) and aprokaryotic selection marker, such as ampicillin resistance, and anorigin of replication for selection and propagation in Escherichia coli.A convenient transfer vector for introducing foreign genes into AcNPV ispAc373. Many other vectors, known to those of skill in the art, havebeen designed. Such a vector is pVL985 (Luckow and Summers, Virology,17:31 (1989)).

A wild-type baculoviral genome and the transfer vector having anexpression cassette insert are transfected into an insect host cellwhere the vector and the wild-type viral genome recombine. Methods forintroducing an expression cassette into a desired site in a baculovirusvirus are known in the art. (Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555, 1987. Smith et al., Mol. Cell.Biol., 3:2156 (1983); and Luckow and Summers, Virology, 17:31 (1989)).For example, the insertion can be into a gene such as the polyhedringene, by homologous double crossover recombination; insertion can alsobe into a restriction enzyme site engineered into the desiredbaculovirus gene (Miller et al., Bioessays, 4:91 (1989)). The expressioncassette, when cloned in place of the polyhedrin gene in the nucleicacid construct, will be flanked both 5′ and 3′ by polyhedrin-specificsequences. An advantage of inserting an expression cassette into thepolyhedrin gene is that occlusion bodies resulting from expression ofthe wild-type polyhedrin gene may be eliminated. This may decreasecontamination of leader proteins produced through expression andformation of occlusion bodies in insect cells by wild-type proteins thatwould otherwise form occlusion bodies in an insect cell having afunctional copy of the polyhedrin gene.

The packaged recombinant virus is expressed and recombinant plaques areidentified and purified. Materials and methods for baculovirus andinsect cell expression systems are commercially available in kit form.(Invitrogen, San Diego, Calif., USA (“MaxBac” kit)). These techniquesare generally known to those skilled in the art and fully described inSummers and Smith, Texas Agricultural Experiment Station Bulletin No.1555, 1987.

Plasmid-based expression systems have also been developed the may beused to introduce an expression cassette of the invention into an insectcell and produce a leader protein. (McCarroll and King, Curr. Opin.Biotechnol., 8:590 (1997)). These plasmids offer an alternative to theproduction of a recombinant virus for the production of leader proteins.

Examples of Vectors Useful in Mammalian Cells

An expression cassette of the invention may be inserted into manymammalian vectors that are known in the art and are commerciallyavailable. (CLONTECH, Carlsbad, Calif.; Promega, Madision, Wis.;Invitrogen, Carlsbad, Calif.). Such vectors may contain additionalelements such as enhancers and introns having functional splice donorand acceptor sites. Nucleic acid constructs may be maintainedextrachromosomally or may integrate in the chromosomal DNA of a hostcell. Mammalian vectors include those derived from animal viruses, whichrequire trans-acting factors to replicate. For example, vectorscontaining the replication systems of papillovaviruses, such as SV40(Gluzman, Cell, 23:175 (1981)) or polyomaviruses, replicate to extremelyhigh copy number in the presence of the appropriate viral T antigen.Additional examples of mammalian vectors include those derived frombovine papillomavirus and Epstein-Barr virus. Additionally, the vectormay have two replication systems, thus allowing it to be maintained, forexample, in mammalian cells for expression and in a prokaryotic host forcloning and amplification. Examples of such mammalian-bacteria shuttlevectors include pMT2 (Kaufman et al., Mol. Cell. Biol., 9:946 (1989))and pHEBO (Shimizu et al., Mol. Cell. Biol., 6:1074 (1986)).

E) Host Cells

Host cells producing the recombinant precursor polypeptides for themethods of the invention include prokaryotic and eukaryotic cells ofsingle and multiple cell organisms. Bacteria, fungi, plant, insect,vertebrate and its subclass mammalian cells and organisms may beemployed. Single cell cultures from such sources as well as functionaltissue and whole organisms can operate as production hosts according tothe invention. Examples include E. coli, tobacco plant culture, maize,soybean, fly larva, mice, rats, hamsters, as well as CHO cell cultures,immortal cell lines and the like.

In a preferred embodiment, bacteria are used as host cells. Examples ofbacteria include, but are not limited to, Gram-negative andGram-positive organisms. Escherichia coli is a preferred organism forexpression of preselected polypeptides and amplification of nucleic acidconstructs. Many publicly available E. coli strains include K-strainssuch as MM294 (ATCC 31, 466); X1776 (ATCC 31, 537); KS 772 (ATCC 53,635); JM109; MC1061; HMS 174; and the B-strain BL21. Recombination minusstrains may be used for nucleic acid construct amplification to avoidrecombination events. Such recombination events may remove concatemersof open reading frames as well as cause inactivation of an expressioncassette. Furthermore, bacterial strains that do not express a selectprotease may also be useful for expression of preselected polypeptidesto reduce proteolytic processing of expressed polypeptides. Such strainsinclude, for example, Y1090hsdR, which is deficient in the lon protease.

Eukaryotic cells may also be used to produce a preselected polypeptideand for amplifying a nucleic acid construct. Eukaryotic cells are usefulfor producing a preselected polypeptide when additional cellularprocessing is desired. For example, a preselected polypeptide may beexpressed in a eukaryotic cell when glycosylation of the polypeptide isdesired. Examples of eukaryotic cell lines that may be used include, butare not limited to: AS52, H187, mouse L cells, NIH-3T3, HeLa, Jurkat,CHO-K1, COS-7, BHK-21, A-431, HEK293, L6, CV-1, HepG2, HC11, MDCK,silkworm cells, mosquito cells, and yeast.

F) Transformation

Methods for introducing exogenous DNA into bacteria are available in theart, and usually include either the transformation of bacteria treatedwith CaCl₂ or other agents, such as divalent cations and DMSO. DNA canalso be introduced into bacterial cells by electroporation, use of abacteriophage, or ballistic transformation. Transformation proceduresusually vary with the bacterial species to be transformed (see, e.g.,Masson et al., FEMS Microbiol. Lett., 60:273, 1989; Palva et al., Proc.Natl. Acad. Sci. USA, 79:5582, 1982; EPO Publ. Nos. 034 259 and 063 953;PCT Publ. No. WO 84/04541 [Bacillus], Miller et al., Proc. Natl. Acad.Sci. USA, 8:856, 1988; Wang et al., J. Bacteriol., 172:949, 1990[Campylobacter], Cohen et al., Proc. Natl. Acad. Sci. USA, 69: 2110,1973; Dower et al., Nuc. Acids Res., 16:6127, 1988; Kushner, “Animproved method for transformation of Escherichia coli withColE1-derived plasmids”, in: Genetic Engineering: Proceedings of theInternational Symposium on Genetic Engineering (eds. H. W. Boyer and S.Nicosia), 1978; Mandel et al., J. Mol. Biol., 53:159, 1970; Taketo,Biochim. Biophys. Acta, 949:318, 1988 [Escherichia], Chassy et al., FEMSMicrobiol. Lett., 44:173, 1987 [Lactobacillus], Fiedler et al., Anal.Biochem, 170:38, 1988 [Pseudomonas], Augustin et al., FEMS Microbiol.Lett., 66:203, 1990 [Staphylococcus], Barany et al., J. Bacteriol.,144:698, 1980; Harlander, “Transformation of Streptococcus lactis byelectroporation”, in: Streptococcal Genetics (ed. J. Ferretti and R.Curtiss III), 1987; Perry et al., Infec. Immun., 32: 1295, 1981; Powellet al., Appl. Environ. Microbiol., 54: 655, 1988; Somkuti et al., Proc.4th Eur. Cong. Biotechnology, 1:412, 1987 [Streptococcus]).

Methods for introducing exogenous DNA into yeast hosts are well-known inthe art, and usually include either the transformation of spheroplastsor of intact yeast cells treated with alkali cations. Transformationprocedures usually vary with the yeast species to be transformed (see,e.g., Kurtz et al., Mol. Cell. Biol., 6:142 (1986); Kunze et al., J.Basic Microbiol., 25:141 (1985) [Candida], Gleeson et al., J. Gen.Microbiol., 132:3459 (1986); Roggenkamp et al., Mol. Gen. Genet.,202:302 (1986) [Hansenula], Das et al., J. Bacteriol., 158:1165 (1984);De Louvencourt et al., J. Bacteriol., 754:737 (1983); Van den Berg etal., Bio/Technology, 8:135 (1990) [Kluyveromyces], Cregg et al., Mol.Cell. Biol., 5:3376 (1985); Kunze et al., J. Basic Microbiol., 25:141(1985); U.S. Pat. Nos. 4,837,148 and 4,929,555 [Pichia], Hinnen et al.,Proc. Natl. Acad. Sci. USA, 75:1929 (1978); Ito et al., J. Bacteriol.,153:163 (1983) [Saccharomyces], Beach and Nurse, Nature, 300:706 (1981)[Schizosaccharomyces], and Davidow et al., Curr. Genet., 10:39 (1985);Gaillardin et al., Curr. Genet., 10:49 (1985) [Yarrowia]).

Exogenous DNA is conveniently introduced into insect cells through useof recombinant viruses, such as the baculoviruses described herein.

Methods for introduction of heterologous polynucleotides into mammaliancells are known in the art and include lipid-mediated transfection,dextran-mediated transfection, calcium phosphate precipitation,polybrene-mediated transfection, protoplast leader, electroporation,encapsulation of the polynucleotide(s) in liposomes, biollistics, anddirect microinjection of the DNA into nuclei. The choice of methoddepends on the cell being transformed as certain transformation methodsare more efficient with one type of cell than another. (Feigner et al.,Proc. Natl. Acad. Sci., 84:7413 (1987); Feigner et al., J. Biol. Chem.,269:2550 (1994); Graham and van der Eb, Virology, 52:456 (1973); Vaheriand Pagano, Virology, 27:434 (1965); Neuman et al., EMBO J., 1:841(1982); Zimmerman, Biochem. Biophys. Acta., 694:227 (1982); Sanford etal., Methods Enzymol., 217:483 (1993); Kawai and Nishizawa, Mol. Cell.Biol., 4:1172 (1984); Chaney et al., Somat. Cell Mol. Genet., 12:237(1986); Aubin et al., Methods Mol. Biol., 62:319 (1997)). In addition,many commercial kits and reagents for transfection of eukaryotic areavailable.

Following transformation or transfection of a nucleic acid into a cell,the cell may be selected for through use of a selectable marker. Aselectable marker is generally encoded on the nucleic acid beingintroduced into the recipient cell. However, co-transfection ofselectable marker can also be used during introduction of nucleic acidinto a host cell. Selectable markers that can be expressed in therecipient host cell may include, but are not limited to, genes whichrender the recipient host cell resistant to drugs such as actinomycinC₁, actinomycin D, amphotericin, ampicillin, bleomycin, carbenicillin,chloramphenicol, geneticin, gentamycin, hygromycin B, kanamycinmonosulfate, methotrexate, mitomycin C, neomycin B sulfate, novobiocinsodium salt, penicillin G sodium salt, puromycin dihydrochloride,rifampicin, streptomycin sulfate, tetracycline hydrochloride, anderythromycin. (Davies et al., Ann. Rev. Microbiol., 32:469, 1978).Selectable markers may also include biosynthetic genes, such as those inthe histidine, tryptophan, and leucine biosynthetic pathways. Upontransfection or transformation of a host cell, the cell is placed intocontact with an appropriate selection marker.

For example, if a bacterium is transformed with a nucleic acid constructthat encodes resistance to ampicillin, the transformed bacterium may beplaced on an agar plate containing ampicillin. Thereafter, cells intowhich the nucleic acid construct was not introduced would be prohibitedfrom growing to produce a colony while colonies would be formed by thosebacteria that were successfully transformed.

EXAMPLES

The following series of Examples illustrates procedures for cloning,expression and detection of precursor polypeptides that can be used togenerate a peptide of interest. Examples 1 through 5 provide theprotocol and experimental procedures used for preparing peptides ofinterest using the clostripain cleavage techniques of the presentinvention. Example 6 provides the application of these protocols andprocedures to specific peptides. The peptides chosen are GLP-2(1-34) andGLP-2(1-33, A2G). Example 7 provides data showing the parameters foraffecting selectivity of the clostripain cleavage of GLP-2(1-34).Example 10 provides the application of these protocols and procedures toGLP-2(1-33, A2G). Example 11 provides data showing the parameters foraffecting selectivity of the clostripain cleavage to GLP-2(1-33,A2G).This series of examples are intended to illustrate certain aspects ofthe invention and are not intended to be limiting thereof.

Example 1 Construction of Vectors that Contain DNA Which Encodes aDesired Precursor Polypeptide

In order to express a desired precursor polypeptide, an expressionvector, pBN121 or pBN122, was constructed through use of PCR,restriction enzyme digestion, DNA ligation, transformation into abacterial host, and screening procedures according to proceduresdescribed, for example, in Sambrook et al., Molecular Cloning (2^(nd)edition). Preferably the vector contains regulatory elements thatprovide for high level expression of a desired precursor polypeptide.Examples of such regulatory elements include, but are not limited to: aninducible promoter such as the chlorella virus promoter (U.S. Pat. No.6,316,224); an origin of replication for maintaining the vector in highcopy number such as a modified pMB1 promoter; a LaqIq gene for promotersuppression; an aminophosphotransferase gene for kanamycin resistance;and a GST terminator for terminating mRNA synthesis (FIGS. 1 and 1A).The pBN121 vector uses a Tac promoter instead of the chlorella viruspromoter.

E. coli is a preferred host. To clone the expression cassette ofT7tag-GSDR-[GLP-2(1-34)]₆(SEQ ID NO:37), or T7tagVg-VDDR-GLP-2(1-33,A2G)(SEQ ID NO:40), PCR or multiple PCR extension was performed tosynthesize DNA encoding the T7tag and the indicated GLP-2 gene usingpreferred codons for E. coli. DNA providing the T7 gene 10 ribosomebinding site and the first twelve amino acids (T7tag) after initiationcodon was cloned into plasmid pBN121 or pBN122 at XbaI-SalI sitesbetween the promoter and the terminator. DNA encoding the hydrophobiccore of the Vestigial (Vg) gene [Williams et al., Genes Dev. Dec.,5:2481 (1991)] was cloned into the plasmid at BamHI-SalI sites. DNAencoding GLP-2(1-33, A2G) or GLP-2(1-34) was cloned into pBN122 orpBN121 respectively at SalI-XhoI sites. Plasmids were transformed intoE. coli using heat shock or electroporation procedures (2^(nd) edition,Sambrook et. al). Cells were streaked onto LB+Kanamycin+agar plates,cultures were grown in LB+Kanamycin media from single colonies. Plasmidsfrom these cultures were prepared, screened by restriction enzymedigestion, and sequenced using DNA sequencers. The cultures with thecorrect plasmid sequence were saved in glycerol stock at −80° C. orbelow.

Alternative peptides can be cloned by this method using differentcombinations of restriction enzymes and restriction sites according tomethods known in the art.

Example 2 Expression of the Precursor Polypeptide

A shaking flask was inoculated from a glycerol stock of an E. colistrain containing a pBN121 or pBN122 plasmid encoding the desiredpolypeptide. A complex media containing 1% tryptone was employed thatwas supplemented with glucose and kanamycin. The shaking culture wasgrown in a rotary shaker at 37° C. until the optical density was 1.5±0.5at 540 nm. The contents of the shaking flask culture were then used toinoculate a 5 L fermentation tank containing a defined minimal mediacontaining magnesium, calcium, phosphate and an assortment of tracemetals. Glucose served as the carbon source. Kanamycin was added tomaintain selection of the recombinant plasmid. During fermentation,dissolved oxygen was controlled at 40% by cascading agitation andareation with additional oxygen. A solution of ammonium hydroxide wasused to control the pH at about pH 6.9.

Cell growth was monitored at 540 nm until a target optical density ofbetween about 75 OD, was reached and isopropyl-β D-thiogalactoside (IPTGat between 0.1 and 1.0 mM) was added to induce expression of the desiredpolypeptide (FIG. 2). When induction was complete, the cells were cooledin the fermenter and harvested with a continuous flow solid bowlcentrifuge. The sedimented cells were frozen until used.

The frozen cell pellet was thawed and homogenized in 50 mM Tris, 2.5 mMEDTA, pH 7.8. Inclusion bodies were washed in water and were collectedby solid bowl centrifugation. Alternatively, cells were suspended in 8Murea then lysed by conventional means and then centrifuged. Thesupernatant fluid contained the precursor peptide.

Example 3 Detection of Precursor Polypeptides

To monitor the production of the GLP-2(1-34) precursor polypeptidepreparation, cell free extract was diluted 5-fold in 0.2 M HCl in 7.2 Murea. A sample of 15 μl was injected into a Waters Symmetry C-18 columnconnected to a LCM spectrophotometer. The sample was eluted with alinear gradient from 20% Buffer B (95% acetonitrile, 0.1% TFA) to 75%Buffer B over 15 minutes. The gradient was then charged from 75% B to100% B over 1 minute. The column was then eluted with 100% Buffer B.Buffer A was 5% acetonitrile with 0.1% TFA.

The precursor polypeptide peak area is compared to the peak area from areference polypeptide standard chromatographed under the sameconditions. The precursor polypeptide concentration (FIG. 3A—Peak 1) isdetermined by normalizing for the different calculated molarabsorptivities (ε_(280 nm)) of a standard and the precursor polypeptide,injection volumes, and dilution factors. Alternatively, the molarabsorbtivity of the precursor peptide can be estimated from theproportional contributions of the molar absorbtivities at 280 nm of theconstituent amino acids. FIG. 3B shows the mass spectrum of Peak 1 ofFIG. 3A. The precursor polypeptide had a molecular weight of 24,963.

To monitor the production of a T7tagVg-VDDR-GLP-2(1-33,A2G) (SEQ IDNO:40), or T7tag-GSDR-GLP-1(1-33)A2G-PGDR-GLP-2(1-33,A2G) (SEQ ID NO:39)precursor polypeptide preparation, 100 μL of sample (fermentationculture or from a purification process step) was dissolved in 1 mL 71%phenol, 0.6 M citric acid, vortexed and bath sonicated briefly. Thedissolved sample was diluted 12.5-fold to 50-fold in 50% acetonitrile,0.09% TFA, and centrifuged to render it compatible with thechromatography system to be employed. The dissolved precursorpolypeptide and E. coli cell products remain soluble in the dilutedsolution, while other insoluble matters are removed.

The samples were then analyzed using a tapered, 5 μm Magic Bullet C4column (Michrom BioResources). The absolute peak area of the precursorpolypeptide was obtained by recording the absorbance at 280 nm as afunction of time. The HPLC method was as follows:

-   -   1. Mobile phase: A—0.1% TFA in water, B—0.08% TFA in        acetonitrile.    -   2. Detection: 280 nm.    -   3. Gradient: 1 mL/min. at 50° C., using 10-90% B(2.5 min.),        90-10% B(0.1 min.), 10% B(1.4 min.). The gradient may be        modified for better separation of different precursor peptides.    -   4. Injection: 1-10 μL.

The precursor polypeptide peak area is compared to the peak area from areference polypeptide standard chromatographed under the sameconditions. The precursor polypeptide concentration is determined bynormalizing for the different calculated molar absorptivities(ε_(280 nm)) of a standard and the precursor polypeptide, injectionvolumes, and dilution factors. Alternatively, the molar absorbtivity ofthe precursor peptide can be estimated from the proportionalcontributions of the molar absorbances E_((280 nm)) of the constituentamino acids. Multiplying the polypeptide concentration times the processstep volume yields the total quantity of polypeptide (FIGS. 3C and 3D).

Example 4 Cleavage of Precursor Polypeptides

GLP-2(1-34) precursor polypeptide: About 100 grams of cells resuspendedin a buffer containing 50 mm Tris (pH 7.5) and 5 mM EDTA werehomogenized in a Ranie high pressure homogenizer to produce a cell freeextract. About 45 milliliters of the resulting cell extract containingabout 445 mg of the T7tag-GSDR-[GLP-2(1-34)]₆ (SEQ ID NO:37) precursorpolypeptide was adjusted to pH 6.4 with about 100 ml of NaH₂PO₄ (100 mM)and the solution was rendered 1 mM CaCl₂ and 1 mM DTT. The digestionreaction was initiated by the addition of 0.1 unit of clostripain permilligram of precursor polypeptide. The solution was incubated at 25° C.for approximately 3 hours. The time course of the digestion is shown inFIG. 4.

GLP-2(1-33,A2G) precursor polypeptide: Approximately 100 grams of E.coli cells containing the desired precursor polypeptide were lysed bycombining them with approximately two liters of 8 M urea containing 0.1M NH₄OH, pH 10.0 (adjusted with reagent grade HCl). This treatmentcaused the cells to lyse and produce a cell free extract. Alternatively,cells can be lysed with 8 M urea at neutral pH. Lysis methods utilizingurea are preferably used to lyse cells that express soluble precursorpolypeptides.

Recombinant clostripain was prepared as 1400 unit/mL solution. Dilutionswere made, when necessary, in 25 mM HEPES buffer at pH 7.1 with 10 mMDTT and 5 mM CaCl₂ and were stored at 4° C. or in an ice bucket beforeuse.

In one example, the lysate was homogenized for 3 minutes using acommercial homogenizer. The suspension was then centrifuged for 45minutes at 16,900×g. The supernatant fluid was diluted to a finalprotein concentration of from 0.1 to 2 mg/ml in 50 mM HEPES buffer,containing 1 mM CaCl₂ and 1 mM cysteine. Alternately the lysate wassubjected to tangential flow filtration (TFF) using an 8 kD exclusionmembrane. The loss in the filtered volume was replaced with 50 mM HEPEScontaining 0-3 M urea, 1 mM CaCl₂, and 1 mM cysteine, pH 6.0-6.9.

For cells that express precursor polypeptides in inclusion bodies, celllysis was preferably performed by sonication or mechanicalhomogenization in 50 mM Tris, 2.5 mM EDTA, pH 7.5. Centrifugation wasthen be performed to sediment the inclusion bodies. After thesupernatant fluid was decanted, the pellet was dissolved in 8 M urea,mechanically homogenized for 2 minutes then centrifuged to remove theinsoluble material. The supernatant fluid was treated as above to reducethe urea concentration.

Enzymatic digestion of the precursor polypeptide was initiated bycombining about 0.01 to 2 U/mg of precursor polypeptide and clostripain.In this example, the reaction contained 0.45 mg/ml of precursorpolypeptide and 0.2 units of recombinant clostripain per mg precursorpolypeptide. The digest was allowed to proceed for up to 3 hours (FIG.4A).

Example 5 Identification of Reactants and Products Following Digestionof a Precursor Polypeptide by Clostripain

The identity of products produced by cleavage of a precursor polypeptideby clostripain was determined by liquid chromatography/mass spectroscopy(LC/MS) analysis. In one example, a cleavage reaction containingclostripain and a T7tag-GSDR-[GLP-2(1-34)]₆ (SEQ ID NO:37) precursorpolypeptide was assembled that contained 3 mg/ml precursor polypeptideand 0.4 units clostripain per mg of precursor peptide. In anotherexample, a cleavage reaction containing clostripain and aT7tag-GSDR-GLP-2(1-33,A2G)-PGDR-GLP-2(1-33,A2G) (SEQ ID NO:39) precursorpolypeptide was assembled that contained 3 mg/ml precursor polypeptideand 0.4 Units clostripain per mg of precursor peptide. The cleavagereactions were conducted for 80 minutes and resulted in a 90% conversionto the indicated products. A 30 μl aliquot was obtained from a cleavagereaction and mixed with 100 μl of a solution containing 8 M urea towhich 20 μl of 0.1 M EDTA (pH 6.5) was added. Samples were clarified bycentrifugation if needed.

Prepared samples (5 μl) were injected into a Finnigan LCQ DUO ion trapmass spectrometer equipped with a Waters Symmetry C18 column operatingin a positive ion electrospray mode for analysis. During the samplingperiod, molecular weight determination was performed by full scan massspectrometry. Typical MS conditions included a scan range of 300-2000Dale.

LC analysis was performed on a system consisting of a Xcaliber software,ThermoQuest Surveyor MS pumps, a ThermoQuest Surveyor UVspectrophotometric PDA detector and a ThermoQuest Surveyor autosampler.The parameters of the chromotagraphic column are indicated below.

Column: Manufacturer: Waters Company Packing support: Symmetry C18Particle size: 3.5 μM Pore size: 100 Å Column size: 2.1 × 150 mm Guardcolumn: 3.5 μm, 2.1 × 10 mmChromatographic conditions were: flow-rate 300 ul/min and buffers A:0.1% TFA, B: acetonitrile, 0.08% TFA. The gradient was from 15% B to 30%B in 3 minutes, to 55% B in 19 minutes, to 90% B in 3 minutes,temperature 50° C. Detection was over the range 210-320 nm on the PDAdetector, Channel A 214 nm, channel B 280 nm. Mass detection was overthe 300-2000 Dale range. All the samples were analyzed on an LCQ-DUO ESImass spectrometer. Usually, the masses observed with significantrelative abundance are the doubly or triply charged ions, i.e.,[M+2H]²⁺/2 or [M+3H]³⁺/3. The complete mass spectrum as a function oftime could be evaluated following the chromatographic procedure throughuse of the system software. This allows for analysis of individual peaksthat eluted from the column.

The results shown in FIG. 5 illustrate that the identity of peptidesproduced in a cleavage reaction can be identified. FIG. 5 shows cleavageat DRH (FIG. 5, yields peptides C and D). The slower cleavage at ARE) ofthe product C, precursor polypeptide T7tag-GSDR-[GLP-2(1-34)]₆ (SEQ IDNO:37) was not detected, thus the reaction went to completion and yieldspeptides A and B. A purified preparation of GLP-2(1-34)A2G was subjectedto complete amino acid sequence analysis which confirmed the structureof this peptide.

FIGS. 5C and 5D also show that cleavage at DRH (FIG. 5C, peaks 2 and 3)is nearly quantitative (90% yield), while the cleavage at ARD (FIG. 5C,peak 1) was minimal. A purified preparation of GLP-2(1-33,A2G) wassubjected to complete amino acid sequence analysis which confirmed thestructure of this peptide.

Example 6

A. Effects of pH on the Digestion of a Precursor Polypeptide byClostripain

The pH was varied in a series of clostripain cleavage reactions usingthe soluble six-copy GLP-2 polypeptide as substrateT7tag-GSDR-GLP-2(1-34)₆. In the first set of reactions, the bufferutilized was varied with the pH of the reaction mixture, as follows:

For pH 6.28: 50 mM of Piperazine-NN′-bis(2-ethanesulfonic acid)(PIPES);

For pH 6.55: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)(HEPES);

For pH 7.50: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)(HEPES);

For pH 7.94: 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonicacid)(CAPSO);

For pH 8.82: 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonicacid)(CAPSO).

The reaction mixture contained 0.33 mg/mL soluble six-copy GLP-2precursor polypeptide in a cleavage reaction containing 5 mM CaCl₂, 10mM DTT, 4.2 units clostripain per mg of precursor polypeptide, and anappropriate buffer at pH 6.28, 6.55, 7.50, 7.94 or 8.82. The reactiontemperature was 20° C. The pH of the cleavage reaction was measured justbefore addition of clostripain to initiate the reaction. Aliquots of thecleavage reaction were removed at selected time intervals (3, 10, 20 and40 minutes) and quenched in a volume of a solution containing 7.2 M ureaand 1.2 M HCl that was three times the volume of the aliquot. Thequenched aliquot was centrifuged before injection into the HPLC. Peptidecleavage products were detected by the HPLC at 214 nm and 280 nm. Asillustrated in FIG. 6, the fastest cleavage velocity was observed at apH range between about 6.0 and about 7.0. However, loss of the GLP-2monomer by continued internal cleavage was minimized by use of buffer atpH 6.5.

The effect of pH may also be studied in a series of clostripain cleavagereactions using the single copy GLP-2 polypeptide as substrateT7tag-GSDR-GLP-2 (1-33)A2G (SEQ ID NO:44). In the first set ofreactions, the buffer utilized may be varied with the pH of the reactionmixture, as follows:

For pH 6.28: 50 mM of Piperazine-NN′-bis(2-ethanesulfonic acid)(PIPES);

For pH 6.55: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)(HEPES);

For pH 7.50: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)(HEPES);

For pH 7.94: 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonicacid)(CAPSO); For pH 8.82:3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid)(CAPSO).

The reaction mixture may contain 0.33 mg/mL T7tag-GSDR-GLP-2 (1-33, A2G)(SEQ ID NO:44) precursor polypeptide in a cleavage reaction containing 5mM CaCl₂, 10 mM DTT, 4.2 units clostripain per mg of precursorpolypeptide, and an appropriate buffer at pH 6.28, 6.55, 7.50, 7.94 or8.82. The reaction temperature may be kept at 20° C. The pH of thecleavage reaction may be measured just before addition of clostripain toinitiate the reaction. Aliquots of the cleavage reaction can be removedat selected time intervals (3, 10, 20 and 40 minutes) and quenched in avolume of a solution containing 7.2 M urea and 1.2 M HCl that can bethree times the volume of the aliquot. The quenched aliquot may becentrifuged before injection into the HPLC. Peptide cleavage productsmay be detected by the HPLC at 214 nm and 280 nm. As illustrated in FIG.6A for the cleavage of a GLP-2 (1-33,A2G) substrate tested in a similarstudy, the fastest cleavage velocity was observed at a pH range betweenabout 6.0 and about 7.0. However, loss of the GLP-2 monomer by continuedinternal cleavage can be minimized by use of buffer at pH 6.0.

B. Influence of Urea on the Cleavage of a Precursor Polypeptide byClostripain

The effect of urea on the cleavage of a precursor polypeptide byclostripain was tested by cleaving a T7tagVg-VDDR-GLP-2(1-33,A2G) (SEQID NO:40) precursor polypeptide in the presence of various ureaconcentrations. The precursor polypeptide (0.4 mg/ml) was cleaved withclostripain (3.3 Units per mg of precursor polypeptide) in a reactionmixture containing 50 mM HEPES buffer (pH 6.3), 1 mM CaCl₂, 1 mMcysteine, and various concentrations of urea at 25° C. The ureaconcentrations tested were 0, 0.5, 1.0 and 1.5 M. Aliquots of thecleavage reaction were removed at one minute intervals for 10 minutesand quenched by addition of EDTA to a final concentration of 10 mM.Peptide cleavage products were analyzed by the HPLC at 214 nm and 280 nmas previously described. As illustrated in FIG. 6B, the fastest cleavagevelocity was observed in the absence of urea. Concentrations of ureaabove 1.5 M caused a decreasing yield to about 20% at 6.5 M urea.

Example 7 The Effect of Precursor Polypeptide and ClostripainConcentration

A. The Effect of Precursor Polypeptide Concentration

The concentration of the soluble six-copy GLP-2 polypeptideT7tag-GSDR-[GLP-2(1-34)]₆ (SEQ ID NO:37) was varied in a series ofcleavage reactions to ascertain how much precursor polypeptide canoptimally be cleaved in a single reaction.

A stock solution of the soluble six-copy GLP-2 polypeptide was preparedin 10 mM Tris, 1 mM EDTA, 5 mM of CaCl₂, pH 8.0 buffer. Aliquots of thesubstrate stock solution were withdrawn and added to various reactionmixtures as needed. In this series of experiments the substrateconcentration was varied within the reaction mixture as follows: 0.6,1.2, 2.4 and 4.28 mg/mL. The buffer utilized was a phosphate-basedbuffer at 150 mM (ionic strength about 0.45 M), pH: 6.60±0.01. Asbefore, 10 mM DTT was utilized in the reaction mixture. The reactiontemperature was 21° C. and was initiated by the addition of clostripain.Hydrolysis was terminated at 25 minutes by the addition of 3 volumes of7.2 M urea in 1.2 M HCl. Products of the reaction were analyzed by HPLCaccording to the procedure described in Example 7. The results of thereaction are shown in FIG. 7A. The yield of GLP-2(1-34) was in excess of90%.

The identity of the peptide product prepared according to the describedmethod was confirmed by amino acid sequence analysis by LC-MS-MS asbeing GLP-2(1-34). It was determined that the product had a mass of3921.9 ([M+3H+]=1308.3 m/z). The designated peak was further fragmentedto yield the MS/MS data contained in Table I. The calculated masses arefrom monoisotopes. The charges of the fragments were also indicated as(M+n H+), where n is the number of additional hydrogen ions.

Table I Observed Mass of Peptides from the LC-MS Chromatogram FragmentsCalc. Mass Obs. Mass Conv. Mass n SEQ ID NOHADGS FSDEM NTILD NLAAR DFINW LIQTK ITDR 3920.9 1308.3 3922.08 3 1 ADGS FSDEM NTILD NLAAR DFINW LIQTK ITDR 3783.84 1262.37 3784.11 3 2  DGS FSDEM NTILD NLAAR DFINW LIQTK ITDR 3712.8 1238.97 3713.91 3 3   GS FSDEM NTILD NLAAR DFINW LIQTK ITDR 3597.77 1200.64 3598.92 3 4      FSDEM NTILD NLAAR DFINW LIQTK ITDR* 3410.72 1138.16 3411.48 3 5       SDEM NTILD NLAAR DFINW LIQTK ITDR* 3263.65 817.22 3264.88 4 6  DGS FSDEM NTILD NLAAR DFINW LIQTK ITDR 3712.8 1857.81 3713.91 2 7   GS FSDEM NTILD NLAAR DFINW LIQTK ITDR 3597.77 1799.95 3598.92 2 8       SDEM NTILD NLAAR DFINW LIQTK ITDR* 3263.65 1634.43 3266.8 2 30         EM NTILD NLAAR DFINW LIQTK ITDR 3104.59 1553.41 3104.82 2 31              ILD NLAAR DFINW LIQTK ITDR 2629.42 1315.68 2629.36 2 32                  NLAAR DFINW LIQTK ITDR 2288.23 1144.92 2287.84 2 33*These peptides had lost a CN₂H₃ fragment (43 au) from an arginine sidechain.B. The Effect of Clostripain Concentration

The effect of clostripain concentration on the cleavage of a precursorpolypeptide was determined by combining clostripain withT7tag-GSDR-[GLP-2(1-34)]₆ (SEQ ID NO:37) at various ratios ofclostripain to the precursor polypeptide in a cleavage reaction. Thetested ratios were 1, 1/2, 1/4, 1/8 units of clostripain per mg ofprecursor polypeptide. The concentration of the precursor polypeptidewas kept constant at 1.2 mg/ml. The cleavage reaction was conducted inbuffer containing 10 mM Tris, 1 mM EDTA, and 5 mM of CaCl₂ (pH 8.0) at21° C. The cleavage reactions were initiated by addition of clostripainto the cleavage reactions. Aliquots were withdrawn at selected timeintervals, quenched, and analyzed by HPLC.

As shown in FIG. 7B, the slowest reaction containing a ratio of 1 unitclostripain per 8 mg of precursor polypeptide was three times slowerthan the fastest reaction containing a ratio of 1 unit clostripain per 1mg of substrate. As shown in FIGS. 7A and 7B, a 180 minute reaction at40° C. containing a ratio of 1 unit clostripain per 20 mg of substratewas approximately equivalent to a 20 minute reaction with 1 unitclostripain per 1 mg of substrate at room temperature. It is noteworthythat a reaction containing only 1 unit clostripain per 100 mg ofsubstrate produced a higher ratio of full length GLP-2(1-34) totruncated GLP-2(21-34). It is also noteworthy that a reaction containing1 unit clostripain per 20 mg of precursor polypeptide at the ambientroom temperature was almost complete at 10 hr and produced lessGLP-2(21-34) than did the same reaction at 40° C. after about 3.5 hr. Ananalytical HPLC of the purified product is shown in FIG. 8.

Example 8 Preparation of Highly Purified GLP-2(1-34)

A solution containing approximately 136 milligrams of GLP-2(I-34) wasprepared according to the methods described in Example 4. This solutionwas applied to an Amberchrom CG-300 column (4.4×8.0 cm, about 121.6 ml)that was equiliberated with buffer A (10% acetonitrile and 5 mM HCl).The sample was loaded at 40 ml/min and washed with buffer A at 40ml/min. The sample was then eluted with a linear gradient of 10% bufferB (70% acetonitrile and 5 mM HCl) to 70% buffer B in 40 minutes at 30mL/min. Fractions containing GLP-2(1-34) were pooled. The yield ofGLP-2(1-34) was approximately 70%.

The pooled fractions were diluted two fold with dionized water and mixedwith solid urea to form a solution having a final urea concentration ofabout 8 M. A solution containing 1 M N-methyl morpholine (NMM) (50 mL)was then added to produce a solution containing NMM at a finalconcentration of 50 mM. The final solution volume was 1050 ml. The pH ofthe solution was adjusted to 8.3 by the addition of HCl to a finalconcentration of 12 mM. The solution was then filtered through a 0.45 μmmembrane filter. This solution was then applied to a Toyopearl superQ-650S column (1.6×11 cm) at a flow rate of 5 ml/min for a period of onehour and 6 mL/min for 2 hours. The column was washed with buffer A (6 Murea, 50 mM NMM (pH 8.3), 12 mM HCl). The sample was then eluted fromthe column by application of a linear gradient of 0-40% buffer B in 37minutes at a flow rate of 6 mL/min. Fractions containing GLP-2(1-34)were pooled and diluted to a protein concentration below 0.3 mg/mL withbuffer A. The yield was approximately 50%.

The pooled fractions were then diluted two fold with dionized water andacetonitrile was added to a final concentration of about 10%. The samplewas then loaded onto a reverse phase HPLC column (Vydac C18) and thecolumn was washed with buffer A (10% acetonitrile and 5 mM HC1) at aflow rate of 10 ml/min. GLP-2(1-34) was then eluted from the column witha linear gradient of buffer B (70% acetonitrile and 5 mM HCl) of 39-46%in 30 minutes and 46% to 100% in 5 minutes. Fractions containingGLP-2(1-34) were pooled and diluted two-fold with deionized water.

The solution of the previous step was applied to a reverse phase HPLCcolumn (Vydac C18) and the column was washed with buffer A (10%acetonitrile and 10 mM HCl) at a flow rate of 10 mL/min. GLP-2(1-34) waseluted with a linear gradient of 39-46% buffer B (70% acetonitrile and10 mM HCl) in 30 minutes and 46-100% buffer B in 5 minutes. The overallyield of GLP-2(1-34) was about 30% FIG. 8.

Example 9 Production of Variant Forms of GLP-2(1-34), GLP-2(1-34,A2G),and Others

The methods described in examples 1-5 can be used to produce nearly anyvariant of a GLP-2(1-34) or GLP-2(1-34,A2G). An example of such avariant includes, but is not limited to, GLP-2(1-34,M10L). For example,an expression construct can be constructed that expresses theT7tag-GSDR-[GLP-2(1-34,M10L]₆ (SEQ ID NO:45) orT7tag-GSDR-[GLP-2(1-34)]₆A2G (SEQ ID NO:46) precursor polypeptideaccording to the method described in Example 1. This precursorpolypeptide can be expressed and detected according to the methodsdescribed in Examples 2 and 3 and then cleaved according to the methodof Example 4. The identification of GLP-2(1-34,M10L) as the cleavageproduct can be conducted according to the methods described in Example5. Accordingly, analogous methods can be used to create peptide productshaving virtually any desired amino acid substitution.

Example 10 Effect of Organic Solvents on the Digestion of a PrecursorPolypeptide by Clostripain

The effect of organic solvents on the cleavage of a precursorpolypeptide by clostripain was tested by cleaving aT7tagVg-VDDR-GLP-2(1-33,A2G) (SEQ ID NO:40) precursor polypeptide in thepresence of various concentrations of ethanol or acetonitrile (FIGS. 9Aand 9B).

In one example, the precursor polypeptide (1.2 mg/ml) was cleaved withclostripain (5.0 Units per mg of precursor polypeptide) in a reactionmixture containing 50 mM HEPES buffer (pH 6.7), 1 mM CaCl₂, 1 mMcysteine, and 4.8 M urea at 25° C. The ethanol concentrations testedwere 10, 20 and 35% ethanol. The reaction was initiated by the additionof clostripain and allowed to proceed for 30 minutes. The reaction wasterminated by the addition of EDTA to a final concentration of 17 mM.The products of the cleavage reaction were resolved by C4 reverse phasechromatography. Briefly, a 40 μl sample containing the cleavage productswas injected into a Vydac C4 protein column and eluted from the columnthrough application of a gradient composed of Buffer A (5% acetonitrileand 0.1% TFA) and Buffer B (95% acetonitrile and 0.1% TFA). Thefollowing gradient was used: time (minutes) 0, % B: 30; time 7.5, % B:50; time 8.5, % B: 70; time 8.6, % B:30; and time 11, % B:30.

FIG. 9A illustrates the elution position of the major products ofdigestion (peak 1: GLP-2(21-33), peak 2: GLP-2(1-33,A2G), peak 3:precursor polypeptide). It can be seen that increasing concentrations ofethanol cause a) an increase in the rate of disappearance of theprecursor polypeptide (peak 3), b) a concomitant increase in the rate ofthe appearance of the product (peak 2), and c) a decrease in theappearance of an undesired product (peak 1) produced by cleavage of asecondary cleavage site within the precursor polypeptide.

FIG. 9B illustrates the effects of ethanol and acetonitrile on thecleavage rate, and the extent of cleavage, of a precursor polypeptide byclostripain. It can be seen from the figure that the presence of ethanolor acetonitrile in the cleavage reaction increases the rate of cleavageof a precursor polypeptide as well as increases the yield of cleavedproduct. Another surprising result is that production of an undesiredproduct produced by cleavage of a second cleavage site within theprecursor polypeptide is decreased at increased ethanol or acetonitrileconcentrations. These results show that the specificity of clostripaincleavage can be influenced by the presence or absence of an organicsolvent in the cleavage reaction. Thus, the discovery that organicsolvents can influence clostripain cleavage rate and specificity be usedin conjunction with the methods to design clostripain cleavage sites, asdisclosed herein, to produce precursor polypeptides that are selectivelycleaved to yield desired products in high yield (in excess of 90%).

The complete amino acid sequence of a purified preparation ofGLP-2(1-33, A2G) prepared according to the above method was determinedto confirm the composition of the peptide product.

Example 11 Production of Gram Quantities of GLP-2(1-33,A2G)

Whole cells (146 g of cells isolated from 1 liter of culture fromfermentation) expressing the T7tagVg-VDDR-GLP-2(1-33,A2G) (SEQ ID NO:40) precursor polypeptide were suspended in 1 liter of buffer containing8M urea, 50 mM Hepes buffer (pH 6.9), and homogenized for 5 minutesusing a hand held homogenizer (Omni 5000). The suspension was thencentrifuged for 30 minutes at 10,000 rpm (Sorvall centrifuge, SLA 3000rotor) to remove cellular debris. The clear supernatant solution (1000ml) was found to contain 5.77 grams of precursor polypeptide byanalytical reverse-phase HPLC.

For digestion of the precursor polypeptide, the 8M urea supernatantsolution containing the precursor polypeptide was diluted with 4 litersof a buffer (40% ethanol:60% 50 mM Hepes buffer (pH 6.9)) and digestedfor 20 minutes at room temperature with recombinant clostripain (about15 units of clostripain per mg precursor peptide). The reaction wasterminated by the addition of 200 ml EDTA (0.25M) and then centrifugedfor 30 minutes to obtain a clear supernatant solution containing 2.1 gof GLP-2(1-33,A2G).

The digestion products were then subjected to anion exchangechromatography. The pH of the digestion reaction containing 2.1 g of thedigested GLP-2(1-33,A2G) was adjusted to 8.5 and then loaded onto acolumn of Toyopearl Super Q 650S (250 ml bed volume) equilibrated with20 mM Tris buffer pH 8.5. Due to the volume of resin available thechromatography was performed in two stages using ˜2.5 liters of digestsolution per load. The column was washed with Tris buffer and theneluted sequentially with NaCl solutions at concentrations of 0.1M, 0.3Mand 2M in the same buffer. The peptide eluted from the resin with 0.3Msalt. Pigmented material and other contaminants were found to elute atboth higher and lower salt concentrations. The yield of GLP-2(1-33,A2G)from this chromatography step was 1.8 g.

The GLP-2(1-33,A2G) prepared by ion-exchange chromatography was loadedonto a column of Amberchrome CG-71 (1 liter bed volume) equilibratedwith 5 mM HCl for reverse phase chromatography. The resin was washedwith 20 mM Tris buffer pH 8.5 and then eluted with 5% ethanol, 5 mM HClfollowed by 40% ethanol, 10% iso-propanol, 5 mM HCl. Approximately 65%of the GLP-2(1-33,A2G) eluted from the column with the 5% ethanolsolution while the remainder appeared to be more tightly bound andeluted with 40% ethanol. The GLP-2(1-33,A2G) prepared in this mannerexhibited a purity of about 97% when analyzed by reverse phase HPLC. Thetotal recovery of GLP-2(1-33,A2G) was 1.1 gram. The GLP-2(1-33,A2G)produced according to this method had the correct mass and amino acidsequence. FIG. 10 illustrates an analytical reverse phase chromatogramof a sample of the purified material under the conditions of theanalysis.

Example 12 Production of Variant Forms of GLP-2(1-33) andGLP-2(1-33,A2G)

The methods described in Examples 1-5 can be used to produce nearly anyvariant of a GLP-2(1-33) or GLP-2(1-33,A2G). An example of such avariant includes, but is not limited to, GLP-2(133,M10L). For example,an expression construct can be constructed that expresses theT7tagVg-VDDR-GLP-2(1-33,M10L) (SEQ ID NO:40) orT7tag-GLP-2(1-33,A2G)-GPDR-GLP-2(1-33,M10L) (SEQ ID NO:47) precursorpolypeptide according to the method described in Example 1. Thisprecursor polypeptide can be expressed and detected according to themethods described in Examples 2 and 3 and then cleaved according to themethod of Example 4. The identification of GLP-2(1-33,M10L) as thecleavage product can be conducted according to the methods described inExample 5. Accordingly, analogous methods can be used to create peptideproducts having virtually any desired amino acid substitution.

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All publications, patents and patent applications cited herein andpriority U.S. patent application Nos. 60/383,359 and 60/383468 areincorporated herein by reference. The foregoing specification has beendescribed in relation to certain embodiments thereof, and many detailshave been set forth for purposes of illustration, however, it will beapparent to those skilled in the art that the invention is susceptibleto additional embodiments and that certain of the details describedherein may be varied considerably without departing from the basicprinciples of the invention.

What is claimed is:
 1. An isolated nucleic acid encoding a polypeptidefor production of a GLP-2 peptide, the encoded polypeptide comprising:two or more copies of the GLP-2 peptide; and a clostripain cleavagerecognition sequence; wherein the clostripain cleavage recognitionsequence is comprised of amino acids at the N-terminus and/or theC-terminus of the GLP-2 peptide and/or of an optional linker, theclostripain cleavage recognition sequence positioned to allow cleavageby clostripain between the copies of the GLP-2 peptide; wherein theclostripain cleavage recognition sequence comprises Formula I:-Xaa₁-Xaa₂-Xaa₃-  (I); wherein Xaa₁ is aspartic acid, glycine, prolineor glutamic acid, Xaa₂ is arginine, and Xaa₃ is not an acidic aminoacid.
 2. The nucleic acid of claim 1, wherein the GLP-2 peptide is anative GLP-2 peptide.
 3. The nucleic acid of claim 2, wherein the GLP-2peptide is GLP-2(1-34) (SEQ ID NO:9).
 4. The nucleic acid of claim 2,wherein the GLP-2 peptide is GLP-2(1-33) (SEQ ID NO:11).
 5. The nucleicacid of claim 1, wherein the GLP-2 peptide is a variant of a nativeGLP-2 peptide.
 6. The nucleic acid of claim 5, wherein the GLP-2 peptideis GLP-2(1-34,A2G) (SEQ ID NO:15).
 7. The nucleic acid of claim 5,wherein the GLP-2 peptide is GLP-2(1-33,A2G) (SEQ ID NO:13).
 8. Thenucleic acid of claim 1, wherein the encoded polypeptide for productionof the GLP-2 peptide comprises the following formula:Tag-Linker-[Peptide]_(q) wherein, Tag comprises SEQ ID NO:17 or 18;Linker comprises at least one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ IDNO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35 andSEQ ID NO: 36; Peptide is GLP-2(1-34) (SEQ ID NO: 9) or GLP-2(1-34, A2G)(SEQ ID NO: 15); and q is an integer of 2 to about
 20. 9. The nucleicacid of claim 1, wherein the encoded polypeptide for production of theGLP-2 peptide comprises the following formula:Tag-Linker-[Peptide-Linker₂]_(q) wherein: Tag comprises SEQ ID NO:17 orSEQ ID NO: 18; Linker comprises at least one of SEQ ID NO: 23, SEQ IDNO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQID NO: 35 and SEQ ID NO: 36; Linker₂ is SEQ ID NO:23; Peptide isGLP-2(1-34) (SEQ ID NO: 9) or GLP-2(1-34, A2G) (SEQ ID NO: 15); and q isan integer of 2 to about
 20. 10. An isolated nucleic acid encoding apolypeptide for production of a GLP-2 peptide, the encoded polypeptidecomprising: a GLP-2 peptide; wherein the GLP-2 peptide is selected fromGLP-2(1-34) (SEQ ID NO:9), GLP-2(1-33) (SEQ ID NO:11), GLP-2(1-34,A2G)(SEQ ID NO:15) and GLP-2(1-33,A2G) (SEQ ID NO:13); and a clostripaincleavage recognition sequence; wherein the clostripain cleavagerecognition sequence is comprised of amino acids at the N-terminus ofthe GLP-2 peptide and/or of an optional linker, the clostripain cleavagerecognition sequence positioned to allow cleavage by clostripain at theN-terminus of the GLP-2 peptide; wherein the clostripain cleavagerecognition sequence comprises Formula I:-Xaa₁-Xaa₂-Xaa₃-  (I); wherein Xaa₁ is aspartic acid, glycine, prolineor glutamic acid, Xaa₂ is arginine, and Xaa₃ is not an acidic aminoacid.
 11. The nucleic acid of claim 10, wherein the GLP-2 peptide is anative GLP-2 peptide.
 12. The nucleic acid of claim 11, wherein theGLP-2 peptide is GLP-2(1-34) (SEQ ID NO:9).
 13. The nucleic acid ofclaim 11, wherein the GLP-2 peptide is GLP-2(1-33) (SEQ ID NO:11). 14.The nucleic acid of claim 10, wherein the GLP-2 peptide is a variant ofa native GLP-2 peptide.
 15. The nucleic acid of claim 14, wherein theGLP-2 peptide is GLP-2(1-34,A2G) (SEQ ID NO:15).
 16. The nucleic acid ofclaim 14, wherein the GLP-2 peptide is GLP-2(1-33,A2G) (SEQ ID NO:13).17. The nucleic acid of claim 10, wherein the encoded polypeptide forproduction of the GLP-2 peptide comprises the following formula:Tag-Linker-Peptide wherein, Tag comprises SEQ ID NO:17 or 18; Linkercomprises at least one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25,SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ IDNO: 36; and Peptide is GLP-2(1-33) (SEQ ID NO:11) or GLP-2(1-33,A2G)(SEQ ID NO:13).
 18. An isolated nucleic acid encoding a polypeptide forproduction of a GLP-2 peptide, the encoded polypeptide comprising thefollowing formula:Xaa₁-Xaa₂-Peptide wherein Xaa₁ is aspartic acid, glycine, proline orglutamic acid, Xaa₂ is arginine, and Peptide is a GLP-2 peptide; andwherein the GLP-2 peptide is selected from GLP-2(1-34) (SEQ ID NO:9,GLP-2(1-33) (SEQ ID NO:11), GLP-2(1-34,A2G) (SEQ ID NO:15) andGLP-2(1-33,A2G) (SEQ ID NO:13).
 19. The nucleic acid of claim 18,wherein the GLP-2 peptide is GLP-2(1-33,A2G).